Biology subjects

Spielmann, M.

Publications and source records attributed to Spielmann, M..

9 recordsLinked to original sources

bioRxiv2026

Sphingolipid-Driven Lipotoxicity Promotes Calcific Aortic Valve Disease Through ER-Stress-NF-κB-EndMT Signaling and Is Therapeutically Targetable Through Serine Palmitoyltransferase Inhibition

Background: Calcific aortic valve disease (CAVD) lacks disease-modifying therapy. LDL-cholesterol lowering does not attenuate valvular calcification, suggesting cholesterol-independent drivers. Thus, we tested whether ceramide accumulation drives CAVD and whether inhibiting sphingolipid de novo synthesis is therapeutic. Methods: We integrated lipidomics and proteomics of calcified and non-calcified human aortic valves with matched plasma. Mechanistic effects of ceramides were tested in primary human valvular interstitial cells (VICs). Therapeutic efficacy of serine palmitoyltransferase (SPT) inhibition with Myriocin was evaluated in ApoE-/- mice with PCSK9DY-AAV-driven hyperlipidemia on Western diet using preventive treatment and therapeutic initiation after echocardiographically verified gradient elevation. Myriocin was additionally tested in a normolipidemic wild-type aortic valve wire-injury model. Hemodynamics, valve structure, lipid composition, and cell-state transitions were assessed by echocardiography, histology, lipidomics, and single-cell RNA sequencing. Results: Calcified human valves showed enrichment of long-chain ceramides linked to NF-{kappa}B, endothelial-to-mesenchymal transition (EndMT), and ER-stress/unfolded protein response (UPR) programs, whereas matched plasma sphingolipids were not elevated. In hVICs, long-chain C18 ceramide induced GRP78/BiP, CHOP, and ATF4, activated NF-{kappa}B, and promoted dose-dependent calcification. In vivo, SPT inhibition with Myriocin lowered valvular ceramides, reduced leaflet inflammation and mineralization, and improved cusp separation and transvalvular hemodynamics without lowering plasma LDL-cholesterol. In a therapeutic protocol initiated after gradient elevation, Myriocin halted further hemodynamic progression, providing the first evidence that de novo sphingolipid synthesis can be targeted after established CAVD onset in vivo. Myriocin also attenuated injury-induced stenosis in normolipidemic mice. Single-cell RNA sequencing showed reduced NF-{kappa}B/EndMT and ER-stress signatures and shifted VIC states away from osteogenic phenotypes. Conclusions: Human valve lipidomics and human VIC experiments identify ceramide-driven lipotoxicity as a driver of an ER-stress-NF-{kappa}B-EndMT axis in CAVD. In vivo, de novo sphingolipid synthesis is an actionable, LDL-independent target capable of modifying established hemodynamic disease progression, supporting SPT inhibition as a tractable strategy for medical CAVD therapy.

molecular biology↗

bioRxiv2026

Single-cell resolution uncovers cell type-specific dysregulation in Parkin-deficient neuron-microglia co-cultures

BackgroundMutations in the E3 ubiquitin ligase Parkin (encoded by PRKN) are the most frequently known cause of recessively inherited Parkinsons disease. In addition to the loss of dopaminergic neurons, microglial activation is another pathological feature observed in Parkinsons disease. While postmortem brain samples show the end stage of the disease, neurons and glia derived from patients induced pluripotent stem cells (iPSCs) provide a model for detecting early pre-degenerative disease trajectories. However, mixed cell populations often confound these cultures, leading to heterogeneous disease phenotypes. MethodsHere, we tease apart the cell type-specific phenotypes underlying Parkin-linked Parkinsons disease by performing single-nucleus RNA sequencing in iPSC-derived co-cultures of dopaminergic neurons and microglia from PRKN mutation carriers and healthy controls. We validated our transcriptomic key findings through inflammatory cytokine profiling and live-cell calcium imaging. ResultsSingle-nucleus RNA sequencing identified seven major cell types composed of neuronal, glial, and precursor cells, with dopaminergic neurons accounting for the largest cell population. Pathway analysis revealed cell type-specific dysregulated biological processes in Parkin-deficient cells, including gene expression differences in dopaminergic neurons that control mitophagy and dopamine homeostasis, whereas microglia showed changes in calcium homeostasis and inflammatory signaling. Functional analysis verified elevated secretion of monocyte chemotactic protein 1 in PRKN-mutant co-cultures compared with controls, linking Parkin deficiency to increased microglial chemotactic signaling. Furthermore, lower intracellular calcium levels and diminished calcium release following treatment confirmed impaired calcium homeostasis in PRKN-mutant microglia. ConclusionsProfiling at the single-cell level resolved distinct cell subpopulations, enabling us to identify cell type-specific pathway disturbances underlying Parkin deficiency. This unique dataset provides a basis for understanding the impairment of individual cell types and the impact of cellular crosstalk in Parkinsons disease pathology.

molecular biology↗

bioRxiv2025

Vascularized midbrain assembloids show neuroinflammation and dopaminergic neuron vulnerability in Parkinsons Disease

The use of micro-physiological systems has rapidly risen in the last years due to their translatability and complex cellular composition. Human midbrain-specific organoids contain neuroectoderm-derived cell types and are suitable for brain region-specific disease modeling. However, the lack of vasculature in these systems reduces oxygenation and nutrient supply. Furthermore, neurovascular interactions cannot be studied, and disease phenotypes affecting vascular and neurovascular structures cannot be assessed. To overcome these limitations, in this work, we successfully incorporated a vascular network into midbrain organoids by fusion with vascular organoids. Midbrain-vascular assembloids are enriched in vascular cells and microglia. We observed a decrease in hypoxia and cell death in these assembloids. Furthermore, microglia and endothelial cells increased their morphological complexity. Assembloids derived from a Parkinsons disease patient carrying a LRRK2-G2019S mutation displayed a pro-inflammatory phenotype and altered electrophysiological properties. Midbrain-vascular assembloids increase the midbrain model complexity and allow for neuroinflammation studies in Parkinsons disease.

neuroscience↗

bioRxiv2024

Astrocytes in the mouse suprachiasmatic nuclei respond directly to glucocorticoids feedback

The circadian timing system anticipates daily recurring changes in the environment to synchronize physiology. In mammals, the master pacemaker is the hypothalamic suprachiasmatic nuclei (SCN), which synchronizes "wake" functions by inducing the circadian release of Glucocorticoids (GCs) from the adrenal gland. GCs peak right before the active phase and set the time of peripheral clocks, however, it is still unclear whether the SCN respond to GCs feedback. While GCs influence directly the SCN during the perinatal period, the adult circuit is considered to be resistant to them, suggesting a reduction of GCs-sensitivity along development. To understand this mechanism, we followed the expression of GC receptor (GR) along mouse SCN development with single-cell resolution and show that GR is up-regulated in astrocytes as the circuit matures. We provide in vivo and in vitro evidence that the adult SCN stays responsive to circulating GCs through the activation of GR in astrocytes. Astrocytes communication is necessary to induce the GC-dependent shift on the SCN clock. Our data provides insight into the development of the SCN and highlight a new role of astrocytes as time-keepers in the adult. This finding might shed light on how the circadian system adapts to jetlag or shift work.

developmental biology↗

bioRxiv2023

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

developmental biology↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve Split-Hand/Foot Malformation type 3

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigated the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice resulted in restructuring of the chromatin architecture. This led to an ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern. Artificial repositioning of the AER-specific enhancers of Fgf8 was sufficient to induce misexpression of Lbx1 and Btrc. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression and dosage in the developing limb. Our results reveal new insights into the molecular mechanism underlying SHFM3 and provide novel conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

genetics↗

bioRxiv2021

Systematic reconstruction of the cellular trajectories of mammalian embryogenesis

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single cell zygote gives rise to millions of cells expressing a panoply of molecular programs, including much of the diversity that will subsequently be present in adult tissues. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here we set out to integrate several single cell RNA-seq datasets (scRNA-seq) that collectively span mouse gastrulation and organogenesis. We define cell states at each of 19 successive stages spanning E3.5 to E13.5, heuristically connect them with their pseudo-ancestors and pseudo-descendants, and for a subset of stages, deconvolve their approximate spatial distributions. Despite being constructed through automated procedures, the resulting trajectories of mammalian embryogenesis (TOME) are largely consistent with our contemporary understanding of mammalian development. We leverage TOME to nominate transcription factors (TF) and TF motifs as key regulators of each branch point at which a new cell type emerges. Finally, to facilitate comparisons across vertebrates, we apply the same procedures to single cell datasets of zebrafish and frog embryogenesis, and nominate "cell type homologs" based on shared regulators and transcriptional states.

developmental biology↗

bioRxiv2020

VarFish - Collaborative and Comprehensive Variant Analysis for Diagnosis and Research

VarFish is a user-friendly web application for the quality control, filtering, prioritization, analysis, and user-based annotation of panel and exome variant data for rare disease genetics. It is capable of processing variant call files with single or multiple samples. The variants are automatically annotated with population frequencies, molecular impact, and presence in databases such as ClinVar. Further, it provides support for pathogenicity scores including CADD, MutationTaster, and phenotypic similarity scores. Users can filter variants based on these annotations and presumed inheritance pattern and sort the results by these scores. Filtered variants are listed with their annotations and many useful link-outs to genome browsers, other gene/variant data portals, and external tools for variant assessment. VarFish allows user to create their own annotations including support for variant assessment following ACMG-AMP guidelines. In close collaboration with medical practitioners, VarFish was designed for variant analysis and prioritization in diagnostic and research settings as described in the softwares extensive manual. The user interface has been optimized for supporting these protocols. Users can install VarFish on their own in-house servers where it provides additional lab notebook features for collaborative analysis and allows re-analysis of cases, e.g., after update of genotype or phenotype databases.

bioinformatics↗