技术资料

Degeneration of neuromuscular synapses is a key pathological feature of spinal muscular atrophy (SMA),yet cellular mechanisms underlying synapse dysfunction remain elusive. Here,we show that pharmacological stimulation with Roscovitine triggers the assembly of Munc13-1 release sites that relies on its local translation. Our findings show that presynaptic mRNA levels and local synthesis of Munc13-1 are diminished in motoneurons from SMA mice and hiPSC-derived motoneurons from SMA patients. Replacement of the Munc13-1 3’UTR with that of Synaptophysin1 rescues Munc13-1 mRNA transport in SMA motoneurons and restores the nanoscale architecture of presynaptic Munc13-1 release sites. Restoration of Munc13-1 levels leads to functional synaptic recovery in cultured SMA motoneurons. Furthermore,SMA mice cross-bred with a conditional knock-in mouse expressing modified Munc13-1 with a heterologous 3’UTR display attenuated synapse and neurodegeneration and improved motor function. Identifying Munc13-1 as an SMA modifier underscores the potential of targeting synapses to mitigate neuromuscular dysfunction in SMA. Defective neurotransmission is a hallmark of spinal muscular atrophy (SMA). Here,the authors show that local presynaptic Munc13-1synthesis is defective in SMA and that modification of the Munc13-1 mRNA rescues presynaptic architecture and excitability. View Publication

The increasing demand for efficient recombinant insulin production necessitates the development of scalable,high-yield,and cost-effective bioprocesses. In this study,we engineered a novel mini-proinsulin (nMPI) with enhanced expression properties by shortening the C-peptide and incorporating specific residue substitutions to eliminate the need for enzymatic cleavage. To optimize its production,we applied a hybrid approach combining microscale high-throughput cultivation using the BioLector microbioreactor and statistical modeling via response surface methodology (RSM). Critical medium components were first screened using Plackett–Burman Design (PBD) and refined through Central Composite Design (CDD),identifying glycerol as the most influential factor for yield. Among the four statistically derived formulations,Scenario III demonstrated the highest productivity in the microscale platform (13.00 g/L) and maintained strong performance upon scale-up to a 3-L bioreactor (11.5 g/L). The optimized medium balanced carbon and nitrogen sources to enhance cell viability and maximize protein expression. This study not only confirms the predictive accuracy and scalability of the hybrid optimization system but also introduces a robust production platform for nMPI that can be translated into industrial settings. The workflow presented here can serve as a model for the development of efficient expression systems for complex recombinant proteins in E. coli. View Publication

Hepatitis E virus (HEV) is a significant human pathogen causing both acute and chronic infections worldwide. The cell-intrinsic antiviral response serves as the initial defense against viruses and has been shown to be activated upon HEV infection. HEV can replicate in the presence of this response,but the underlying mechanisms remain poorly understood. Here,we investigated the roles of the structural proteins ORF2 and ORF3 in the cell-intrinsic antiviral response to HEV infection. Mechanistically,we validated that ectopic ORF2,but not ORF3,interfered with antiviral and inflammatory signaling downstream of pattern recognition receptors,in part through interaction with the central adaptor protein TANK binding kinase 1. In the full-length viral context,ORF2 contributed to a reduced antiviral response and consequently,more efficient viral replication. In addition,we discovered a protective mechanism mediated by ORF2 that shielded viral replication from antiviral effectors. Using single-cell RNA-sequencing,we confirmed that the presence of ORF2 in infected cells dampened antiviral responses in both actively infected cells and bystanders. As a consequence,we found that early in the infection process,the progression of authentic HEV infection relied on the presence of ORF2,facilitating a balance between viral replication and the antiviral response. Altogether,our findings shed new light on the multifaceted role of ORF2 in the HEV life cycle and improve our understanding of the determinants that contribute to persistent HEV replication in cell culture. Author summaryHepatitis E virus (HEV) is an important yet often underestimated pathogen. Depending on the genotype,HEV infections can progress to chronicity,but the underlying mechanisms remain poorly understood. To gain insight into potential determinants,we investigated how HEV evades the cell-intrinsic antiviral response. We discovered that the HEV capsid protein ORF2 is crucial in limiting this response by interfering with antiviral signaling pathways and shielding viral replication from immune effectors. This balance between viral replication and the antiviral response contributes to persistent HEV infection in cell culture. Our findings reveal a new role for the HEV capsid protein in the viral life cycle and highlight it as an important target for novel therapeutic approaches. View Publication

Alpha-synuclein (aSyn) post-translational modifications (PTM),especially phosphorylation at serine 129 and C-terminal truncations,are highly enriched in Lewy bodies (LB),Lewy neurites,and other pathological aggregates in Parkinson’s disease and synucleinopathies. However,the precise role of these PTM in pathology formation,neurodegeneration,and pathology spreading remains unclear. Here,we systematically investigated the role of post-fibrillization C-terminal aSyn truncations in regulating uptake,processing,seeding,and LB-like inclusion formation using a neuronal seeding model that recapitulates LB formation and neurodegeneration. We show that C-terminal cleavage of aSyn fibrils occurs rapidly post exogenous fibril internalization and during intracellular LB-like inclusion formation. Blocking cleavage of internalized fibrils does not affect seeding,but inhibiting enzymes such as calpains 1 and 2 alters LB-like inclusion formation. We show that C-terminal truncations,along with other PTMs,regulate fibril interactome remodeling,shortening,lateral association,and packing. These findings reveal distinct roles of C-terminal truncations at different aggregation stages on the pathway to LB formation,highlighting the need for consideration of stage‑specific strategies to target aSyn proteolytic cleavages. View Publication

Brain organoids derived from human pluripotent stem cells (hPSCs) hold immense potential for modeling neurodevelopmental processes and disorders. However,their experimental variability and undefined organoid selection criteria for analysis hinder reproducibility. As part of the Bavarian ForInter consortium,we generated 72 brain organoids from distinct hPSC lines. We conducted a comprehensive analysis of their morphological and cellular characteristics at an early stage of their development. In our assessment,the Feret diameter emerged as a reliable,single parameter that characterizes brain organoid quality. Transcriptomic analysis of our organoid identified the abundance of unintended mesodermal differentiation as a major confounder of unguided brain organoid differentiation,correlating with Feret diameter. High-quality organoids consistently displayed a lower presence of mesenchymal cells. These findings provide a framework for enhancing brain organoid standardization and reproducibility,underscoring the need for morphological quality controls and considering the influence of mesenchymal cells on organoid-based modeling. Subject terms: Mesenchymal stem cells,Induced pluripotent stem cells,Stem-cell differentiation View Publication

Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication

INTRODUCTIONPolygenic risk score (PRS) identifies individuals at high genetic risk for Alzheimer's disease (AD),but its utility in predicting cognitive trajectories and AD pathologies remains unclear. We optimized PRS (optPRS) for AD,investigated its association with cognitive trajectories and AD phenotypes of cerebral organoids.METHODSUsing genome‐wide association study (GWAS) summary statistics from a European population,we developed optPRS to predict AD in Korean individuals (n = 1634). We analyzed the association between optPRS and cognitive trajectories (n = 771). We generated induced pluripotent stem cell–derived cerebral organoids from patients with high (n = 3) and low (n = 4) optPRS to evaluate amyloid beta (Aβ) and phosphorylated tau (p‐tau) levels.RESULTSOptPRS predicted AD dementia and Aβ positivity,independent of apolipoprotein E (APOE). Higher optPRSs correlated with rapid cognitive decline. Cerebral organoids from the high optPRS group exhibited increased Aβ insolubility and p‐tau levels.CONCLUSIONOptPRS predicted cognitive decline and AD phenotypes of cerebral organoids,supporting its use in risk assessments and drug‐screening platform.Highlights Optimized polygenic risk scores (optPRSs) improve the prediction of Alzheimer's disease (AD) dementia and amyloid beta positivity (Aβ+).High optPRS is associated with faster cognitive decline,particularly in Aβ+.Induced pluripotent stem cell (iPSC)–derived cerebral organoids from high optPRSs show high Aβ insolubility and phosphorylated tau (p‐tau).PRS genetic risk stratification provides insight into AD progression and pathology. View Publication

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however,its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here,we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids,revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation,expressed SOX2,and lacked CRX,consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas,particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities,demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1,Flavopiridol) and the cell-cycle inhibitor Volasertib,indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively,our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma,establishes a robust and clinically relevant human retinal organoid platform,and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype. View Publication