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Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases,including BRCA1/2 mutant breast cancer,responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing,we identify a TAM_C3 cluster,expressing genes implicated in anti-inflammatory activity,that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity,respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus,targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies. PARP inhibitors (PARPi) have been approved for the treatment of metastatic triple-negative breast cancer (BC),however resistance and recurrence are often observed. Here,in preclinical models of BRCA1/2 wild type and homologous recombination competent BC,the authors show that C5aR1-positive tumor associated macrophages are associated with PARPi-resistance,suggesting targeting C5aR1 as a therapeutic option. View Publication

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS),accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions,differentiated these to functional motor and cortical neurons and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons,decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis,reduced levels of the anti-apoptotic protein Bcl-2,increased endoplasmic reticulum (ER) stress and reduced mitochondrial membrane potential. Furthermore,C9orf72 motor neurons,and also cortical neurons,show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats,which describes a novel pathogenic link between C9orf72 mutations,dysregulation of calcium signalling and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia (FTD). This article is protected by copyright. All rights reserved. View Publication

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream,playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases,including amyotrophic lateral sclerosis (ALS); however,the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression,compromised barrier integrity,and increased P-glycoprotein transporter activity. In addition,mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover,our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally,C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00528-6. View Publication

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The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation,but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA,AMPK,and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase,AMPK,also prevented NF-kappaB activation. Further,we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis. View Publication

CACNA1A encodes the pore-forming α 1A subunit of the Ca V 2.1 calcium channel,whose altered function is associated with various neurological disorders,including forms of ataxia,epilepsy,and migraine. In this study,we generated isogenic iPSC-derived neural cultures carrying CACNA1A loss-of-function mutations differently affecting Ca V 2.1 splice isoforms. Morphological,molecular,and functional analyses revealed an essential role of CACNA1A in neurodevelopmental processes. We found that different CACNA1A loss-of-function mutations produce distinct neurodevelopmental deficits. The F1491S mutation,which is located in a constitutive domain of the channel and therefore causes a complete loss-of-function,impaired neural induction at very early stages,as demonstrated by changes in single-cell transcriptomic signatures of neural progenitors,and by defective polarization of neurons. By contrast,cells carrying the Y1854X mutation,which selectively impacts the synaptically-expressed Ca V 2.1[EFa] isoform,behaved normally in terms of neural induction but showed altered neuronal network composition and lack of synchronized activity. Our findings reveal previously unrecognized roles of CACNA1A in the mechanisms underlying neural induction and neural network dynamics and highlight the differential contribution of the divergent variants Ca V 2.1[EFa] and Ca V 2.1[EFb] in the development of human neuronal cells. The online version contains supplementary material available at 10.1007/s00018-025-05740-7. View Publication

The family of calcium activated potassium channels of low and intermediate conductance,known as SK channels,consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes,such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date,the role of SK channels in developmental processes has been merely a marginal focus of investigation,although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently,several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein,we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells,neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly,we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis. ?? 2013 Elsevier GmbH. View Publication

RATIONALE: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis,the key process underlying excitation-contraction coupling. OBJECTIVE: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). METHODS AND RESULTS: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes,spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore,in contrast with hESC-derived cardiomyocytes,ryanodine did not reduce the amplitudes,maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition,spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2),sacroplasmic recticulum calcium-ATPase (SERCA),junction (Jun) and triadin (TRDN),were significantly lower in hiPSC than in hESCs. CONCLUSIONS: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. View Publication

Ribosome profiling,which is based on deep sequencing of ribosome footprints,has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However,the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here,we overcome these hurdles through the development of “Ribo-FilterOut”,which is based on the separation of footprints from ribosome subunits by ultrafiltration,and “Ribo-Calibration”,which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript,the translation initiation rate,and the overall number of translation events before its decay,all in a genome-wide manner. Moreover,our method reveals the allocation of ribosomes under heat shock stress,during aging,and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome. Ribosome profiling faces issues with rRNA contamination and measurements of ribosome numbers on transcripts. Here,the authors develop Ribo-FilterOut and Ribo-Calibration,methods which can be used to estimate kinetic and stoichiometric parameters of translation under various conditions. View Publication

Clinical translation of tissue-engineered advanced therapeutic medicinal products is hindered by a lack of patient-dependent and independent in-process biological quality controls that are reflective of in vivo outcomes. Recent insights into the mechanism of native bone repair highlight a robust path dependence. Organoid-based bottom-up developmental engineering mimics this path-dependence to design personalized living implants scaffold-free,with in-build outcome predictability. Yet,adequate (noninvasive) quality metrics of engineered tissues are lacking. Moreover,insufficient insight into the role of donor variability and biological sex as influencing factors for the mechanism toward bone repair hinders the implementation of such protocols for personalized bone implants. Here,male and female bone-forming organoids were compared to non-bone-forming organoids regarding their extracellular matrix composition,transcriptome,and secreted proteome signatures to directly link in vivo outcomes to quality metrics. As a result,donor variability in bone-forming callus organoids pointed towards two distinct pathways to bone,through either a hypertrophic cartilage or a fibrocartilaginous template. The followed pathway was determined early,as a biological sex-dependent activation of distinct progenitor populations. Independent of donor or biological sex,a cartilage-to-bone transition was driven by a common panel of secreted factors that played a role in extracellular matrix remodeling,mineralization,and attraction of vasculature. Hence,the secreted proteome is a source of noninvasive biomarkers that report on biological potency and could be the missing link toward data-driven decision-making in organoid-based bone tissue engineering. Subject terms: Bone,Bone quality and biomechanics View Publication