Philipp Mergenthaler

Mitochondrial programmed cell death (i.e. intrinsic apoptosis) is regulated through the formation of pro-apoptotic protein complexes at the mitochondrial outer membrane (MOM), which permeabilize the MOM and commit the cell to die. This step is modulated by mitochondrial dynamics. To capture this, we treated cells with the mitochondrial dynamics inhibitor M1 before inducing apoptosis with actinomycin D (ActD), and imaged them by high-throughput spinning-disk confocal microscopy. In mouse embryonic fibroblasts (MEFs) we followed apoptosis by long-term live-cell imaging, and in human induced pluripotent stem cells (hiPSCs) we stained fixed cells for the pro-apoptotc protein BAX at mitochondria. Here, we provide a description of these two large-scale microscopy datasets. Dataset 1 contains 637,632 images of MEFs imaged as time series after labelling of mitochondrial morphology and integrity with the mitochondrial membrane potential-sensitive dye tetramethylrhodamine-ethylester (TMRE) and apoptosis with Annexin V-FITC. The dataset also contains a digital phase contrast channel which can be useful to track cells. Dataset 2 contains 9,504 images of hiPSCs with immunofluorescence labelling of BAX and COX IV. The datasets are made available through the BioImage Archive repository.

BAX is a critical regulator of the apoptosis cascade and its abnormal activation contributes to the development of disease including cardiovascular and neurodegenerative disorders. Therefore, BAX represents an attractive target for the pharmacological inhibition of pathological cell death. Based on a virtual screen, we identified and modified several EGFR kinase inhibitors with potential activity against BAX. Systematic modifications of the dacomitinib and gefitinib core structure were performed, allowing us to elucidate how structural modifications influence biological activity and leading to a comprehensive structure-activity relationship (SAR) on BAX inhibition. The developed series has favorable drug-like properties and improved BAX inhibitory activity in vitro, in cells, and in human induced pluripotent stem cell-derived brain organoids, with significantly reduced EGFR kinase activity. This series therefore represents a promising starting point for the development of improved and potentially therapeutic BAX inhibitors.

Summary: Speckle artifacts and isolated foreground pixels are common in fluorescence microscopy and can interfere with segmentation and subsequent quantitative image analysis. Conventional denoising methods often modify image intensities through filtering or smoothing, potentially altering biologically relevant fluorescence signals. We introduce Sparse Pixel Cluster Cleaning (SPC-Clean), a topology-aware method that removes poorly supported foreground pixels through iterative neighborhood analysis of a thresholded mask. SPC-Clean is deterministic, training-free, preserves original fluorescence intensities for practical microscopy workflows. Availability and implementation: SPC-Clean is available as an open-source Python Napari plugin for microscopy image processing. Source code, documentation, and example data are available at: https://github.com/MergenthalerLab/SPC-Clean.git . Contact: pendar.alirezazadeh@charite.de or philipp.mergenthaler@charite.de

INTRODUCTION: Myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS) are autoimmune diseases of the neuromuscular junction resulting in fatigable muscle weakness. Rituximab (RTX) is used to treat patients refractory to standard immunosuppression, but evidence for its efficacy remains inconsistent. Here, we analyzed real-world data on the clinical course and side effects of RTX in MG and LEMS patients. METHODS: This was a single-center study of all patients diagnosed with MG (n=64) or LEMS (n=5) treated with RTX from 2011 until 2021. Outcomes of RTX treatment were recorded retrospectively with Myasthenia Gravis Foundation of America Post-Intervention Status (MGFA-PIS), number of rescue therapies, myasthenic crises, and steroid dose at 1-year and 2-year follow-ups. RESULTS: MGFA-PIS improved at both 1-year (y) and 2-y follow-up compared with baseline. Incidence rates of rescue therapies per 100 person-months (95% CI) decreased from 15.0 (11.8–18.8) at baseline to 7.5 (4.7–12.3) at 1-y and 4.3 (2.5–7.8) at 2y-follow-up. The number of patients without myasthenic crises within one year increased from baseline (49, 86.0%) to 1y-follow-up (55, 96.5%). Median (IQR) daily steroid dose decreased from 10 (5– 22.5) mg/d at baseline to 4 (0–10) mg/d at 1y-follow-up, and to 2.5 (0–10) mg/d at 2y-follow-up. CONCLUSION: This study indicates that RTX was associated with a stabilized clinical course and decreased steroid use in patients with autoimmune myasthenic syndromes, including those with thymoma-associated MG. Our data suggest that therapeutic benefit is apparent within the first year of treatment and is maintained through two years.

ObjectivesLipoprotein receptor-related protein 4 antibody-positive myasthenia gravis (LRP4-Ab-positive MG) is a rare serologic subgroup with poorly defined clinical phenotype. This study investigat...

Background The ischemic penumbra, a metabolically compromised yet potentially salvageable region surrounding the ischemic core, is a prime target for acute stroke intervention. Yet an objective molecular definition of the penumbra, particularly during the earliest stages of ischemia, remains lacking. Methods and Results We applied principal component analysis (PCA) followed by k-means clustering to high-resolution mass spectrometry imaging data covering multiple metabolic pathways to identify a metabolically defined penumbra in a mouse model of hyperacute stroke (45 min middle cerebral artery occlusion, MCAO). Targeted spatial metabolomic profiling by matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) reveals a distinct penumbral metabolic profile, marked by relative preservation of high-energy phosphates, comparable lactate accumulation, and reduced succinate accumulation relative to the core. Spatial transcriptomics revealed selective induction of immediate-early genes, including Npas4, Fos and Junb, within the penumbra. Consistently, imaging mass cytometry shows enrichment of phospho-histone H3 (pHH3) within the penumbra, suggesting a chromatin-associated response potentially linked to immediate-early gene activation. Conclusion Together, these findings provide a multimodal molecular atlas of the hyperacute metabolically defined penumbra and reveal molecular features that facilitates its identification and inform future therapeutic strategies.

Thyroid hormones (THs) are essential regulators of human brain development, and disrupted TH availability during pregnancy or early life is linked to adverse neurodevelopmental outcomes. Concerns that environmental chemicals interfere with TH signalling have increased the need for human-relevant in vitro systems to identify thyroid hormone system-disrupting chemicals (THSDCs) for risk assessment. Here, we compared two human-induced pluripotent stem cell (hiPSC)-derived brain organoid models for THSDC assessment: (i) human cortical organoids (COs) generated by unguided differentiation, offering higher architectural complexity but lower throughput; and (ii) neural stem cell-derived organoids (NSCOs), designed for scalability with reduced cellular diversity. Both models expressed key TH handling components, including the transporter SLC16A2 (MCT8) and the inactivating enzyme DIO3. Using LC–MS/MS, we show that exogenous T3 is depleted from culture media and metabolized to 3,3′-T2 and 3′-T1 in both models, alongside upregulation of T3-responsive genes (HR, KLF9, DIO3, SEMA3C). Pulse and chronic co-exposures to reference disruptors iopanoic acid (IA, deiodinase inhibitor) and silychristin (SC, MCT8 inhibitor) altered T3 metabolism and modulated T3-responsive transcriptional endpoints. In NSCOs, high-content imaging revealed treatment-associated changes in cell composition, with chronic T3 reducing the SOX2-positive progenitor pool and THSDCs blocking this effect. Together, these findings provide a framework for organoid qualification—linking TH handling, transcriptomic responsiveness, and scalable phenotypic readouts—as a necessary step toward model validation and implementation of brain organoids in THSDC risk assessment pipelines.

Introduction: Generalized myasthenia gravis (gMG) may progress to life‐threatening myasthenic crises (MC) requiring mechanical ventilation. Standard therapy includes intravenous immunoglobulins (IVIg), plasmapheresis (PLEX), immunoadsorption (IA), and high‐dose corticosteroids. This study aimed to evaluate the real‐world effectiveness of complement‐inhibitors (C5‐I) in patients refractory to the standard treatment of MC (termed therapy‐refractory MC). Methods: This multicentre, retrospective study included patients with acetylcholine‐receptor‐antibody positive (AChR‐ab+) gMG experiencing MC or severe exacerbations (MGFA IVb/V) treated with eculizumab or ravulizumab in the intensive or intermediate care unit (ICU/IMC). The primary outcome was the proportion of patients discharged from ICU/IMC within six weeks after C5‐I initiation (German Clinical Trials Registry; DRKS00032104). Results: Among 17 identified cases, seven had thymoma‐associated MG (TAMG) and 10 had late‐onset MG; median age was 77 years. Five patients required non‐invasive and 12 invasive ventilation; nine underwent tracheotomy. Therapies included IVIg, PLEX, IA, and corticosteroids. Rituximab was added in two cases. Twelve patients received eculizumab and five ravulizumab. Median hospitalization before C5‐I initiation was 32 days (IQR 22–50) and median ICU‐stay was 17 days (IQR 4.5–35) thereafter. Fourteen patients (82%) reached the primary outcome. Two patients died due to bacterial sepsis; no meningococcal infection was observed. Conclusion: In MC or severe exacerbations insufficiently responsive to standard treatment with IVIg and PLEX/IA, add‐on C5‐I therapy may represent an effective therapeutic approach. Importantly, TAMG, a subtype typically excluded from interventional trials yet often requiring more intensive therapy, also demonstrated clinical improvement. These findings warrant further systematic evaluation of C5‐I as adjunctive therapy for AChR‐ab+ therapy‐refractory MC.

Magnetic resonance imaging (MRI) of glucose metabolism shows significant potential for identifying disease biomarkers and monitoring therapeutic responses in neurological conditions. Here, we present a protocol utilizing chemical exchange-sensitive spin-lock (CESL) MRI with the glucose analogue 2-deoxy-D-glucose (2DG) in the rat brain. We employed this method to characterize metabolic changes in ischemic tissue in a rat model of stroke. However, the utility of the technique is not limited to stroke and may be adapted to other disease models with minimal modifications. Previous research has demonstrated that CESL MRI is sensitive to various glucose analogs, including regular D-glucose, which is suitable for human application. Consequently, our protocol provides a foundation for a wide range of future applications in both basic and translational research, with potential utility in animal models and, eventually, human studies.