Max-Delbrück-Centrum für Molekulare Medizin

Background A lack of serotonin (also known as 5-hydroxytryptamine, 5-HT) in the brain due to deficiency of the rate-limiting enzyme in 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), was recently reported to result in impaired maternal affiliation across species, including mice, rats, and monkeys. In rodents, this was reflected in a lack of preference for maternal odors and reduced levels of isolation-induced ultrasonic vocalizations (USV), possibly contributing to a severe growth retardation phenotype. Methods Here, we tested whether growth retardation, maternal affiliation deficits, and/or impairments in socio-affective communication caused by Tph2 deficiency can be rescued through early social enrichment in rats. To this aim, we compared male and female Tph2−/− knockout and Tph2+/− heterozygous rat pups to Tph2+/+ wildtype littermate controls, with litters being randomly assigned to standard nesting (SN; one mother with her litter) or communal nesting (CN; two mothers with their two litters). Results Our results show that Tph2 deficiency causes severe growth retardation, together with moderate impairments in somatosensory reflexes and thermoregulatory capabilities, partially aggravated by CN. Tph2 deficiency further led to deficits in socio-affective communication, as evidenced by reduced emission of isolation-induced USV, associated with changes in acoustic features, clustering of subtypes, and temporal organization. Although CN did not rescue the impairments in socio-affective communication, CN ameliorated the maternal affiliation deficit caused by Tph2 deficiency in the homing test. To close the communicative loop between mother and pup, we assessed maternal preference and showed that mothers display a preference for Tph2+/+ controls over Tph2−/− pups, particularly under CN conditions. This is consistent with the aggravated growth phenotype in Tph2−/− pups exposed to the more competitive CN environment. Conclusion Together, this indicates that CN aggravates growth retardation despite ameliorating maternal affiliation deficits in Tph2-deficient rat pups, possibly due to reduced and acoustically altered isolation-induced USV, hindering efficient socio-affective communication between mother and pup.

The long-term consequences of cancer and its therapy on the patients’ immune system years after cancer-free survival remain poorly understood. Here, we present an in-depth characterization of the bone marrow immune ecosystem of multiple myeloma long-term survivors, from initial diagnosis up to 17 years following a single therapy line and cancer-free survival. Using comparative single-cell analyses combined with molecular, genomic, and functional approaches, we demonstrate that multiple myeloma long-term survivors exhibit pronounced alterations in their bone marrow microenvironment associated with impaired immunity. These immunological alterations were frequently linked to an inflammatory immune circuit fueled by the long-term persistence or resurgence of residual myeloma cells. Notably, even in the complete absence of any detectable residual disease for decades, sustained changes in the immune system were observed, suggesting an irreversible ‘immunological scarring’ caused by the initial exposure to the cancer and therapy. Collectively, our study provides key insights into the molecular and cellular bone marrow ecosystem of long-term survivors of multiple myeloma, revealing both reversible and irreversible alterations in the immune compartment.

Background The β-adrenergic augmentation of cardiac contraction, by increasing the conductivity of L-type voltage-gated CaV1.2 channels, is of great physiological and pathophysiological importance. Stimulation of β-adrenergic receptors (βAR) activates protein kinase A (PKA) through separation of regulatory (PKAR) from catalytic (PKAC) subunits. Free PKAC phosphorylates the inhibitory protein Rad, leading to increased Ca²⁺ influx. In cardiomyocytes, the core subunit of CaV1.2, CaV1.2α1, exists in two forms: full-length or truncated (lacking the distal C-terminus (dCT)). Signaling efficiency is believed to emanate from protein interactions within multimolecular complexes, such as anchoring PKA (via PKAR) to CaV1.2α1 by A-kinase anchoring proteins (AKAPs). However, AKAPs are inessential for βAR regulation of CaV1.2 in heterologous models, and their role in cardiomyocytes also remains unclear. Results We show that PKAC interacts with CaV1.2α1 in heart and a heterologous model, independently of Rad, PKAR, or AKAPs. Studies with peptide array assays and purified recombinant proteins demonstrate direct binding of PKAC to two domains in CaV1.2α1-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Data indicate both partial competition and possible simultaneous interaction of PCRD and DCRD with PKAC. The βAR regulation of CaV1.2α1 lacking dCT (which harbors DCRD) was preserved, but subtly altered, in a heterologous model, the Xenopus oocyte. Conclusions We discover direct interactions between PKAC and two domains in CaV1.2α1. We propose that these tripartite interactions, if present in vivo, may participate in organizing the multimolecular signaling complex and fine-tuning the βAR effect in cardiomyocytes.

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs can be formed in a clathrin-dependent manner. This process maintains complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV ( 1 ). However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here, we used cryoelectron tomography to visualize molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram averaging, and showed them forming a complex with the membrane-embedded protein synaptophysin (Syp). Our bioluminescence assay revealed pairwise interactions between vesicle-associated membrane protein 2 and Syp and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle size. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with an icosahedral symmetry. We observed V-ATPases under clathrin cages in several isolated clathrin-coated vesicles (CCVs). Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their and CCVs preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs' diversity and their molecular architecture.

Aims Traditional cardiovascular (CV) biomarkers (high‐sensitivity troponinT [hsTnT] and N‐terminal pro‐B‐type natriuretic peptide [NT‐proBNP]) are important to monitor cancer patients' cardiac function and to assess prognosis. Newer CV biomarkers (mid‐regional pro‐adrenomedullin [MR‐proADM], C‐terminal pro‐arginine vasopressin [copeptin], and mid‐regional pro‐atrial natriuretic peptide [MR‐proANP]) might outperform traditional biomarkers. Methods and results Overall, 442 hospitalized cancer patients without significant CV disease or current infection were enrolled (61 ± 15 years, 52% male, advanced cancer stage: 85%) and concentrations of CV biomarkers were analysed. Differences in echocardiographic, clinical, laboratory parameters were assessed. Patients were followed for up to 69 months for all‐cause mortality. In univariable analyses, MR‐proADM, hsTnT, copeptin, MR‐proANP, and NT‐proBNP predicted all‐cause mortality. In multivariable analyses (adjusted for sex, age, Eastern Cooperative Oncology Group performance status, estimated glomerular filtration rate [eGFR], C‐reactive protein, anti‐cancer therapy, reason for hospitalization, cancer stage and type), only MR‐proADM remained an independent predictor of mortality (MR‐proADM per 1 ln: hazard ratio [HR] 2.27, 95% confidence interval [CI] 1.47–3.50], p < 0.001). MR‐proADM had the highest area under the curve (AUC) using receiver operating characteristic analysis (AUC [95% CI] 0.74 [0.69–0.79]; hsTnT: AUC 0.69; copeptin: AUC 0.66; MR‐proANP: AUC 0.63; NT‐proBNP: AUC 0.62). Optimal cut‐point for mortality prediction with MR‐proADM was 0.94 nmol/L (HR 2.43 [95% CI 1.92–3.06], p < 0.001). Patients with MR‐proADM >0.94 nmol/L were older, more often had cancer stage IV, showed reduced performance status, eGFR, haemoglobin, diastolic left ventricular function, and elevated systolic pulmonary artery pressure. Conclusion MR‐proADM is an independent predictor of mortality in advanced stage, hospitalized cancer patients without significant CV disease or current infection. The optimal MR‐proADM cut‐point for mortality prediction was 0.94 nmol/L with hazards for mortality being approximately 2.5 times higher. There was a continuous increase in mortality risk with stepwise increase of MR‐proADM concentrations. Elevated concentrations of MR‐proADM were also associated with reduced performance status and mildly reduced left ventricular diastolic function as well as higher age and more often cancer stage IV.

Chromosomal instability is a major driver of intratumoral heterogeneity (ITH), promoting tumor progression. In the present study, we combined structural variant discovery and nucleosome occupancy profiling with transcriptomic and immunophenotypic changes in single cells to study ITH in complex karyotype acute myeloid leukemia (CK-AML). We observed complex structural variant landscapes within individual cells of patients with CK-AML characterized by linear and circular breakage–fusion–bridge cycles and chromothripsis. We identified three clonal evolution patterns in diagnosis or salvage CK-AML (monoclonal, linear and branched polyclonal), with 75% harboring multiple subclones that frequently displayed ongoing karyotype remodeling. Using patient-derived xenografts, we demonstrated varied clonal evolution of leukemic stem cells (LSCs) and further dissected subclone-specific drug–response profiles to identify LSC-targeting therapies, including BCL-xL inhibition. In paired longitudinal patient samples, we further revealed genetic evolution and cell-type plasticity as mechanisms of disease progression. By dissecting dynamic genomic, phenotypic and functional complexity of CK-AML, our findings offer clinically relevant avenues for characterizing and targeting disease-driving LSCs.

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation.

The asymmetric distribution of RNA within a cell plays a pivotal biological role, ensuring the distinctive shapes and functionality of subcellular compartments. In neurons, these mechanisms are fundamental to cellular growth, synaptic plasticity, and information processing. To understand these mechanisms, diverse methods have been developed to analyze localized transcripts. Here, we outline our optimized method for measurement of mRNA half-lives in subcellular neuronal compartments—neurites, and cytoplasmic and nuclear fractions of cell bodies. We call this method spatial SLAMseq, as it combines SLAMseq with subcellular compartment separation techniques. Spatial SLAMseq facilitates the concurrent measurement of mRNA dynamics and steady-state RNA levels within neuronal subcellular compartments.

Perfusion changes in white matter (WM) lesions and normal‐appearing brain regions play an important pathophysiological role in multiple sclerosis (MS). However, most perfusion imaging methods require exogenous contrast agents, the repeated use of which is discouraged. Using resting‐state functional MRI (rs‐fMRI), we aimed to investigate differences in perfusion between white matter lesions and normal‐appearing brain regions in MS and healthy participants. A total of 41 MS patients and 41 age‐ and sex‐matched healthy participants received rs‐fMRI, from which measures of cerebral hemodynamics and oxygenation were extracted and compared across brain regions and study groups using within‐ and between‐group nonparametric tests, linear mixed models, and robust multiple linear regression. We found longer blood arrival times and lower blood volumes in lesions than in normal‐appearing WM. Higher blood volumes were found in MS patients' deep WM lesions compared to healthy participants, and blood arrival time was more delayed in MS patients' deep WM lesions than in healthy participants. Delayed blood arrival time in the cortical grey matter was associated with greater cognitive impairment in MS patients. Perfusion imaging using rs‐fMRI is useful for WM lesion characterization. rs‐fMRI‐based blood arrival times and volumes are associated with cognitive function.

The aim of the present study was to assess systemic circulatory and tissue activities of both the classical arm and of the alternative arm of the renin-angiotensin system (RAS) in a new transgenic rat line (TG7371) that expresses angiotensin-(1-7) (ANG 1-7)-producing fusion protein; the results were compared with the activities measured in control transgene-negative Hannover Sprague-Dawley (HanSD) rats. Plasma and tissue concentrations of angiotensin II (ANG II) and ANG 1-7, and kidney mRNA expressions of receptors responsible for biological actions of ANG II and ANG 1-7 [i.e. ANG II type 1 and type 2 (AT 1 and AT 2 ) and Mas receptors] were assessed in TG7371 transgene-positive and in HanSD rats. We found that male TG7371 transgene-positive rats exhibited significantly elevated plasma, kidney, heart and lung ANG 1-7 concentrations as compared with control male HanSD rats; by contrast, there was no significant difference in ANG II concentrations and no significant differences in mRNA expression of AT 1 , AT 2 and Mas receptors. In addition, we found that in male TG7371 transgene-positive rats blood pressure was lower than in male HanSD rats. These data indicate that the balance between the classical arm and the alternative arm of the RAS was in male TGR7371 transgene-positive rats markedly shifted in favor of the latter. In conclusion, TG7371 transgene-positive rats represent a new powerful tool to study the long-term role of the alternative arm of the RAS in the pathophysiology and potentially in the treatment of cardio-renal diseases.

Beyond obesity, visceral adipose tissue (VAT) has emerged as an important predictor of chronic disease, but the role of diet quality patterns (DQP) in VAT development is not well defined. Therefore, we conducted a systematic review of how various DQP are associated with VAT via literature searches in PubMed and EMBASE. We included observational investigations in disease-free adults/adolescents that related DQP to VAT assessed by imaging methods. The studies were evaluated separately for a priori and a posteriori DQP and according to design differences. Study quality was assessed using the Risk of Bias in Non-randomised Studies of Interventions tool. Of the 1807 screened articles, thirty-five studies met the inclusion criteria. The majority of a priori indices, for example, the Healthy Eating Index, showed significant inverse associations with VAT, while only a small proportion of a posteriori patterns were related to VAT. Results did not differ substantially by the method of exposure and outcome assessment or between studies with (n 20) or without (n 15) body-size adjustment, but significant findings were more common in younger v. older individuals, USA v. other populations and investigations with moderate v. serious risk of bias. The heterogeneity of the existing literature limited the ability to quantify the magnitude of the associations across studies. These findings suggest that a high-quality diet, as assessed by a priori DQP, is generally inversely associated with VAT, but results for a posteriori DQP are less consistent. As associations persisted after adjusting for body size, diet quality may beneficially influence VAT beyond its association with obesity.

Background The 5’ untranslated region of mRNA strongly impacts the rate of translation initiation. A recent convolutional neural network (CNN) model accurately quantifies the relationship between massively parallel synthetic 5’ untranslated regions (5’UTRs) and translation levels. However, the underlying biological features, which drive model predictions, remain elusive. Uncovering sequence determinants predictive of translation output may allow us to develop a more detailed understanding of translation regulation at the 5’UTR. Results Applying model interpretation, we extract representations of regulatory logic from CNNs trained on synthetic and human 5’UTR reporter data. We reveal a complex interplay of regulatory sequence elements, such as initiation context and upstream open reading frames (uORFs) to influence model predictions. We show that models trained on synthetic data alone do not sufficiently explain translation regulation via the 5’UTR due to differences in the frequency of regulatory motifs compared to natural 5’UTRs. Conclusions Our study demonstrates the significance of model interpretation in understanding model behavior, properties of experimental data and ultimately mRNA translation. By combining synthetic and human 5’UTR reporter data, we develop a model (OptMRL) which better captures the characteristics of human translation regulation. This approach provides a general strategy for building more successful sequence-based models of gene regulation, as it combines global sampling of random sequences with the subspace of naturally occurring sequences. Ultimately, this will enhance our understanding of 5’UTR sequences in disease and our ability to engineer translation output.

Experimental research has uncovered lipocalin 2 (LCN2) as a novel biomarker implicated in the modulation of intestinal inflammation, metabolic homeostasis, and colon carcinogenesis. However, evidence from human research has been scant. We, therefore, explored the association of pre‐diagnostic circulating LCN2 concentrations with incident colorectal cancer (CRC) in a nested case–control study within the in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. LCN2 was measured in 1267 incident CRC cases matched to 1267 controls using incidence density sampling. Conditional logistic regression was used to estimate incidence rate ratios (IRRs) and 95% confidence intervals (95% CIs) according to tumor subsite and sex. Weighted Cox proportional hazard regression was used to explore associations by adiposity status. In multivariable‐adjusted analyses, the IRR [95% CI] per doubling in LCN2 concentration was 1.16 [0.98–1.37] for CRC overall, 1.26 [1.00–1.59] for colon cancer, and 1.08 [0.85–1.38] for rectal cancer. The association for colon cancer was more pronounced in women (IRR [95% CI], 1.66 [1.20–2.30]) and for proximal colon cancer (IRR [95% CI], 1.96 [1.15–3.34]), whereas no association was seen in men and distal colon cancer. The association for colon cancer was positive in individuals with high waist circumference (hazard ratio [95% CI], 1.69 [1.52–1.88]) and inverse in individuals with low waist circumference (hazard ratio [95% CI], 0.86 [0.76–0.98], P interaction<0.01). Overall, these data suggest that pre‐diagnostic LCN2 concentrations were positively associated with colon cancer, particularly occurring in the proximal colon, in women and among individuals with abdominal adiposity.

Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool in the assessment of cardiac structure, morphology, and function. CMR also affords myocardial tissue characterization and probing of cardiac physiology, both being the focus of ongoing research. These developments are fueled by the move to ultrahigh magnetic field strengths, which permit enhanced sensitivity and spatial resolution that help to overcome the limitations of current clinical MR systems. This chapter reviews the potential of using CMR as a means to assess physiology in the heart muscle by exploiting quantification of myocardial effective transverse relaxation times (T2*) for a better understanding of myocardial (patho)physiology. For this purpose, the basic principles of T2* mapping, the biophysical mechanisms governing T2*, and (pre)clinical applications of myocardial T2* mapping are presented. Technological challenges and solutions for T2*-sensitized CMR at ultrahigh magnetic field strengths are discussed followed by a survey of acquisition techniques and post-processing approaches. Preliminary results derived from myocardial T2* mapping of healthy subjects and in patients at 7.0 Tesla are presented. A concluding section provides an outlook including future developments and potential applications.

The chromosomal theory of inheritance dictates that genes on the same chromosome segregate together while genes on different chromosomes assort independently¹. Extrachromosomal DNAs (ecDNAs) are common in cancer and drive oncogene amplification, dysregulated gene expression and intratumoural heterogeneity through random segregation during cell division2,3. Distinct ecDNA sequences, termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells⁴. How multiple ecDNA species within a tumour cell are assorted and maintained across somatic cell generations is unclear. Here we show that cooperative ecDNA species are coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. ecDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy-number gains in multiple ecDNA species before any selection. Intermolecular proximity and active transcription at the start of mitosis facilitate the coordinated segregation of ecDNA species, and transcription inhibition reduces co-segregation. Computational modelling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

Background Incomplete attack remission is the main cause of disability in myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). Apheresis therapies such as plasma exchange and immunoadsorption are widely used in neuroimmunology. Data on apheresis outcomes in MOGAD attacks remain limited. Methods We retrospectively evaluated all apheresis treated attacks occurring in patients with MOGAD between 2008 and 2023 at 18 Neuromyelitis Optica Study Group centres. Treatment response was categorised as complete, partial or no remission. Preattack and follow-up Expanded Disability Status Scale (EDSS) and visual Functional System Scores (FSS) were used to calculate absolute outcomes (ΔEDSS/Δvisual FSS). Predictors of complete remission were analysed using a generalised linear mixed model. Results Apheresis was used for 117/571 (20.5%) attacks in 85/209 (40.7%) patients. Attacks with simultaneous optic neuritis and myelitis were treated more often with apheresis (42.4%, n=14) than isolated myelitis (25.2%, n=35), cerebral manifestation (21.0%, n=17) or isolated optic neuritis (17.6%, n=51). Apheresis was initiated as first-line therapy in 12% (4.5 (IQR 0–11) days after attack onset), second-line therapy in 62% (15 (IQR 6.75–31) days) and third-line therapy in 26% (30 (IQR 19–42) days). Complete remission was achieved in 21%, partial remission in 70% and no remission in 9% of patients. First-line apheresis (OR 2.5, p=0.040) and concomitant disease-modifying therapy (OR 1.5, p=0.011) were associated with complete remission. Both parameters were also associated with a favourable ΔEDSS. No differences in outcomes were observed between the apheresis types. Conclusion Apheresis is frequently used in MOGAD attacks. An early start as first-line therapy and concomitant disease-modifying therapy predict full attack recovery.