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

Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Idorn et al. characterized a mouse strain harboring a mutation identified in an HSE patient. Defective IFN-driven antiviral responses led to hyperactivation of inflammatory responses,which contributed to disease development. The study identifies immunopathology as an important contributor to HSE pathogenesis. 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

The rare and rapidly progressive neurodegenerative disease multiple system atrophy (MSA) mainly affects the striatum and other subcortical brain regions. In this atypical Parkinsonian syndrome,the protein alpha-synuclein aggregates and misfolds in neurons as well as glial cells and is released in elevated amounts by hypoexcitable neurons. Mitochondrial dysregulation affects the biosynthesis of coenzyme Q10 and the activity of the respiratory chain,as shown in an induced pluripotent stem cell (iPSC) model. Proteome studies of cerebrospinal fluid and brain tissue from MSA patients yielded inconsistent results regarding possible protein changes due to small and combined groups of atypical Parkinsonian syndromes. In this study,we analysed the cellular proteome of MSA patient-derived striatal GABAergic medium spiny neurons. We observed 25 significantly upregulated and 16 significantly downregulated proteins in MSA cell lines compared to matched healthy controls. Various protein types involved in diverse molecular functions and cellular processes emphasise the multifaceted pathomechanisms of MSA. These data could contribute to the development of novel disease-modifying treatment strategies for MSA patients. 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

Variants of phospholipase C gamma 2 (PLCG2),a key microglial immune signaling protein,are genetically linked to Alzheimer's disease (AD) risk. Understanding how PLCG2 variants alter microglial function is critical for identifying mechanisms that drive neurodegeneration or resiliency in AD. Induced pluripotent stem cell (iPSC) –derived microglia carrying the protective PLCG2 P522R or risk‐conferring PLCG2 M28L variants,or loss of PLCG2,were generated to ascertain the impact on microglial transcriptome and function. Protective PLCG2 P522R microglia showed significant transcriptomic similarity to isogenic controls. In contrast,risk‐conferring PLCG2 M28L microglia shared similarities with PLCG2 KO microglia,with functionally reduced TREM2 expression,blunted inflammatory responses,and increased proliferation and cell death. Uniquely,PLCG2 P522R microglia showed elevated cytokine secretion after lipopolysaccharide (LPS) stimulation and were protected from apoptosis. These findings demonstrate that PLCG2 variants drive distinct microglia transcriptomes that influence microglial functional responses that could contribute to AD risk and protection. Targeting PLCG2‐mediated signaling may represent a powerful therapeutic strategy to modulate neuroinflammation. The impact of Alzheimer's disease protective‐ and risk‐associated variants of phospholipase C gamma 2 (PLCG2) on the transcriptome and function of induced pluripotent stem cell (iPSC) –derived microglia was investigated. PLCG2 risk variant microglia exhibited a basal transcriptional profile similar to PLCG2‐deficient microglia but significantly different from isotype control and the transcriptionally similar PLCG2 protective variant microglia. PLCG2 risk variant and PLCG2‐deficient microglia show decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2). The differential transcriptional pathways of protective and risk‐associated PLCG2 variant microglia functionally affect proliferation,apoptosis,and immune response. Protective PLCG2 microglia show resilience to apoptosis and increased cytokine/chemokine secretion upon exposure to lipopolysaccharide (LPS). View Publication

Understanding diseases as the result of continuous transitions from a healthy system is more realistic than understanding them as discrete states. Here,we designed the spectrum formation approach (SFA),a machine learning-based method that extracts key features contributing to disease state continuity. We applied the SFA to transcriptomic data from patients with progressive liver disease and neurodegenerative movement disorders to examine its effectiveness in identifying biologically relevant gene sets. The SFA identified transcription factors that potentially regulate liver inflammation and voluntary movement. In neurodegenerative disorders,the SFA also identified genes regulated by ETS-1,with unclear effects on movement. In functional assessment using human iPSC-derived neurons,ETS-1 overexpression disrupted dopamine receptor balance,reduced GABA-producing enzyme levels,and promoted cell death. These findings suggest that the SFA enables the discovery of regulatory factors capable of modifying disease states and provides a framework for the continuity-based interpretation of biological systems. Subject terms: Diseases,Pathogenesis,Signs and symptoms 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