Biology subjects

Engelhardt, S.

Publications and source records attributed to Engelhardt, S..

15 recordsLinked to original sources

bioRxiv2026

The Role of Secondary Chordae Tendineae in Mitral Valve Mechanics: A Benchtop Study

PurposeSecondary chordae tendineae (CT) are frequently disregarded during mitral valve (MV) repair. However, their contribution to MV biomechanics remains poorly understood. We hypothesized that secondary CT play an important role in maintaining leaflet geometry during valve closure. MethodsEleven explanted porcine MVs were micro-CT scanned in a systolic configuration under three conditions: (1) physiological baseline with all secondary CT intact, (2) transection of the A2 strut CT, and (3) transection of all secondary CT. Billowing height (BH), coaptation height (CH) along the parasternal long-axis view, and coaptation area (CA) were quantified and compared between conditions. An increase in BH[≥] 2 mm from baseline was defined as pathological. ResultsTransection of the A2 strut CT increased BH by 3.1 mm, exceeding the pathological billowing threshold by 1.1 mm (padj = 0.003). CH decreased by 0.6 mm (padj = 0.003), whereas CA showed no significant change compared with baseline. Subsequent transection of all secondary CT further increased BH to 4.8 mm relative to baseline, corresponding to 2.8 mm above the billowing threshold (padj = 0.003). CH did not differ significantly, while CA decreased by 0.35 cm2 compared with baseline (padj = 0.029). All values represent medians. ConclusionSecondary CT play a critical role in stabilizing MV leaflet geometry. Their transection resulted in increased leaflet billowing and reduced coaptation metrics associated with unfavorable MV repair outcomes. Preservation or reconstruction of secondary CT may therefore be an important consideration in MV repair strategies.

bioengineering↗

bioRxiv2025

Allele-specific genomics decodes gene targets and mechanisms of the non-coding genome

A large proportion of disease variants is found in non-coding RNAs (ncRNAs), gene loci that have been identified as key regulatory elements. However, for most ncRNAs, their targets are unknown, hindering our ability to understand complex diseases. Here, we found that allele-specific ncRNAs were enriched nearby allelic protein-coding genes (pcGenes), suggesting that the allele-specific information could be used to predict cis-acting ncRNA-targets. We translated this concept into the Allelome.LINK framework and applied it to the major mouse organs, revealing 397 events where the allele-specific expression (ASE) of a ncRNA correlated with the ASE of a nearby pcGene, suggesting either enhancing or repressive regulatory interactions. Next, we applied our strategy to the largest human dataset including tissues from nearly 1,000 individuals. Given the outbred nature of humans, each individual allows for the discovery of novel ASE correlation events. We uncovered 2,291 shared ASE events along with their mechanisms, which we benchmarked against sample-matched eQTLs, yielding a high validation rate of 77.47%. Further GWAS integration assigned 30.59% of variants overlapping informative ncRNAs to their pcGene targets. As more sequencing data and risk variants become available, this strategy has the potential to decode the entire cis-acting landscape of the non-coding genome.

genomics↗

bioRxiv2024

Barley resistance and susceptibility to fungal cell entry involve the interplay of ROP signaling with phosphatidylinositol-monophosphates

Rho-of-plant small GTPases (ROPs) are regulators of plant polar growth and of plant-pathogen interactions. The barley ROP, RACB, is involved in susceptibility towards infection by the barley powdery mildew fungus Blumeria hordei (Bh) but little is known about the cellular pathways that connect RACB-signaling to disease susceptibility. Here we identify novel RACB interaction partners of plant or fungal origin by untargeted co-immunoprecipitation of constitutively active (CA) RACB tagged by green-fluorescent protein from Bh-infected barley epidermal layers and subsequent analysis by liquid chromatography-coupled mass spectrometry. Three of immunoprecipitated proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase and a putative Bh-effector protein are involved in the barley-Bh-pathosystem and support disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in case of RACB, this lipid-interaction is mediated by its carboxy-terminal polybasic region (PBR). Fluorescent markers for anionic phospholipids show altered subcellular distribution in barley cells during Bh-attack and under expression of a RACB-binding fungal effector. Phosphatidylinositol 4-phosphate, phosphatidylinositol 3,5-bisphosphate and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. The interplay of ROPs with anionic phospholipids and phospholipid-metabolizing enzymes may thus enable the subcellular enrichment of components pivotal for success or failure of fungal penetration.

plant biology↗

bioRxiv2024

A retrograde transit filter mediated by optineurin controls mitostasis in distal axons

The mechanisms of mitochondrial homeostasis in neurons-- mitostasis--are enigmatic and disease-prone, given a neurons large synaptic pool of mitochondria. To better understand mitostasis in mature mouse motor axons, we used ex vivo optical pulse-chase measurements of mitochondrial volume flux and identified a reiterative degradation system near presynaptic terminals and distal paranodes, which accounts for 75% of mitochondrial turnover in synapses. This distal filter system captures dysfunctional mitochondria from the retrograde stream and redirects them for lysosomal degradation. It depends on optineurin, a motor neuron disease-related mitophagy adaptor, but not on PINK1 and parkin, implicating a non-canonical mitophagy pathway. In presymptomatic motor neuron disease models, where retrograde transport is disrupted, the fraction of removed mitochondria is increased. Thus, we identify a new mitostasis system in distal axons with a cascade of checkpoints for local mitophagy, which normally maintains mitochondrial mass balance but is disrupted early in degenerative axonopathies.

cell biology↗

bioRxiv2024

Polyglutamylation of microtubules drives neuronal remodeling

Developmental remodeling shapes neural circuits via activity-dependent pruning of synapses and axons. The cytoskeleton is critical for this process, as microtubule loss via enzymatic severing is an early step of pruning across many circuits and species. However, how microtubule-severing enzymes, such as spastin, are activated in specific neuronal compartments remains unknown. Here, we reveal that polyglutamylation, a posttranslational tubulin modification that is enriched in neurons, plays an instructive role in developmental remodeling by tagging microtubules for severing. Motor neuron-specific gene deletion of enzymes that add or remove tubulin polyglutamylation--TTLL glutamylases vs. CCP deglutamylases--accelerates or delays neuromuscular synapse remodeling in a neurotransmission-dependent manner. This mechanism is not specific to peripheral synapses but also operates in central circuits, e.g., the hippocampus. Thus, tubulin polyglutamylation acts as an activity-dependent rheostat of remodeling and shapes neuronal morphology and connectivity.

cell biology↗

bioRxiv2024

Enhanced legumain activity links progranulin deficiency to TDP-43 pathology in frontotemporal lobar degeneration

Loss-of-function mutations in GRN are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43-positive inclusions. Progranulin (PGRN) loss leads to lysosomal dysfunction, microglial hyperactivation, and TDP-43 deposition, yet the underlying pathomechanism remains unknown. We demonstrate that PGRN slows the maturation and limits the proteolytic activity of the lysosomal protease legumain (LGMN). Accordingly, LGMN activity is strongly elevated in Grn knockout (ko) mice, in human induced pluripotent stem cell-derived GRN ko microglia, and in FTLD-GRN patients brain. Secreted microglial LGMN is internalized by neurons, where it mediates pathological processing of TDP-43, which is prevented by selective LGMN inhibition. In contrast, AAV-mediated overexpression of LGMN in mouse brains promotes TDP-43 processing, the aggregation of phosphorylated TDP-43 and increases plasma neurofilament light chain (NfL), a marker for neuronal loss. Our findings identify LGMN as a link between PGRN haploinsufficiency and TDP-43 pathology in FTLD-GRN and suggest LGMN as a therapeutic target.

neuroscience↗

bioRxiv2023

In vivo silencing of regulatory elements using a single AAV-CRISPRi vector

CRISPR-Cas9 based transcriptional repressors (CRISPRi) have emerged as specific and robust tools for functional epigenetic silencing of regulatory elements. Adeno-associated viruses (AAVs) are promising CRISPRi delivery vectors for cardiovascular research and therapy. However, compact vectors enabling codelivery of all CRISPRi components by a single AAV are needed for an enhanced and consistent performance. We engineered a 4.7kb all-in-one CRISPRi construct compatible with AAV-mediated delivery and produced cardiotropic AAVi 6 and 9 particles for in vitro and in vivo application, respectively. AAVi vectors targeting the Nppa promoter (AAViNppa) reduced gene expression in cultivated cardiomyocytes (HL-1 cells) in a dose-dependent manner. The maximum effect was a >95% reduction as measured by qPCR and RNA-seq. This effect was orchestrated by loss of chromatin accessibility (ATAC-seq) and establishment of heterochromatin (H3K9me3 ChIP-seq) specifically at the target promoter region. We confirmed the broad applicability of AAVi to different cardiomyocyte cell culture systems by silencing several genes in primary neonatal rat ventricular cardiomyocytes (NRVMs), human iPSC-derived cardioids and iPSC-CMs. To demonstrate the efficacy of AAVi in vivo we injected 8-week-old C57Bl/6 WT mice with a single dose of AAViNppa and implanted osmotic minipumps releasing Phenylephrine (50 mg/kg/d) and Angiotensin II (0.45 mg/kg/d) to induce Nppa transcription. AAViNppa silenced Nppa transcription as revealed by qPCR and single nuclei RNA-seq even under stress conditions. On the epigenome layer AAViNppa induced closed chromatin at the Nppa promoter site comparable to the in vitro effect. Here, we present an efficient AAV-based method for CRISPRi-mediated epigenetic silencing of gene expression in cardiac myocytes in vivo and in vitro. This functional epigenetic approach provides an efficient way to modulate gene expression in the heart and could become a standard method for cardiovascular disease modelling and translational research.

genomics↗

bioRxiv2023

Gene Therapy for Cardiomyopathy associated with Duchenne Muscular Dystrophy in a Pig Model

BackgroundGenetic cardiomyopathies caused by mutations in the dystrophin gene (DMD) are only partially responsive to current pharmacological heart failure treatments, although dilated and arrhythomogenic phenotypes of cardiomyopathy are frequent. ObjectiveIn this study, we tested whether a normalization of Ca2+-handling by forced expression of SERCA2a in cardiomyocytes mitigates heart failure and arrhythmogenesis in a pig model for Duchenne muscular dystrophy (DMD). Methods and resultsMale offspring of pigs lacking DMD exon 52 are characterized by heart failure with reduced ejection fraction (HFrEF, EF 34.5{+/-}1.8% vs. 49.2{+/-}1.0% in control hearts), arrhythmogenesis due to large apical regions of reduced voltage amplitude and sudden cardiac death with a lifespan of usually less than 4 months. Slow antegrade intracoronary infusion of AAV1.SERCA2a (3x1013 virus genomes (vg) per pig) improved left ventricular ejection fraction (EF 47.3{+/-}2.0%, p<0.05) to a similar extent as germline editing of DMD{Delta}52 to DMD{Delta}51-52, inducing a Becker dystrophy (BMD) genotype (EF 46.7{+/-}3.8%). Moreover, AAV.SERCA2a significantly reduced myocardial inflammation and fibrosis and areas of reduced AP amplitude. ConclusionsIn DMD pigs, 3x1013vg/heart of GMP-grade AAV1.SERCA2a sufficed to normalize left ventricular function and improved electrical vulnerability of the heart. Hence, AAV.SERCA2a may serve as a treatment option for DMD cardiomyopathy.

physiology↗

bioRxiv2023

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodelling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodelling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodelling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/539799v4_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14fa4bcorg.highwire.dtl.DTLVardef@1c86bbborg.highwire.dtl.DTLVardef@118edfcorg.highwire.dtl.DTLVardef@1b06916_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

The lncRNA landscape of cardiac resident macrophages and identification of Schlafenlnc as a regulator of macrophage migratory function

Cardiac resident macrophages (crMPs) were recently shown to exert pivotal functions in cardiac homeostasis and disease, but the underlying molecular mechanisms are largely unclear. Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulatory molecules in a number of cell types, but neither the identity nor the molecular mechanisms of lncRNAs in crMPs are known. Here, we have employed deep RNA-seq and single cell RNA sequencing to resolve the crMP lncRNA landscape from healthy and diseased murine myocardium. CrMPs express previously unknown and highly cell type-specific lncRNAs, among which one lncRNA, termed Schlafenlnc, was particularly abundant and enriched in crMPs. We found Schlafenlnc to be necessary for migration-associated gene expression in macrophages in vitro and in vivo and essential for their adhesion and migration. Collectively, our data provide a basis to the systematic characterization of lncRNAs in crMPs and establish Schlafenlnc as a critical regulator of macrophage migratory functions.

cell biology↗

bioRxiv2022

Plasmacytoid dendritic cells regulate megakaryocyte and platelet homeostasis

Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN- to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-.

immunology↗

bioRxiv2021

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f.sp. hordei

In barley (Hordeum vulgare), the function of ROPs appears central to polar cell development and the interaction outcome with parasitic fungi but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP with GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant specific ROP nucleotide exchanger domain (PRONE). We performed phylogenetic analyses and annotated barley PRONE GEFs. HvGEF14 is expressed in leaf epidermal tissue and downregulated after inoculation with Blumeria graminis f.sp. hordei. Protein-protein interaction assays indicate that HvGEF14 and the type I barley ROP protein HvRACB can interact in yeast and in planta. Overexpression of HvGEF14 further recruited the ROP-GTP downstream interactor HvRIC171 to the cell periphery and let to interaction of HvRACB with an extended CRIB (Cdc42/Rac Interactive Binding motif) peptide of HvRIC171 in a similar manner as constitutively activated HvRACB. Finally, the over expression of HvGEF14 led to enhanced susceptibility to fungal entry while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells, which is crucial for fungal penetration success. HighlightThe activated GTPase HvRACB is a susceptibility factor of barley in response to Blumeria graminis f.sp. hordei. The newly discovered guanine nucleotide exchange factor HvGEF14 is a HvRACB activator.

plant biology↗

bioRxiv2020

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

plant biology↗

bioRxiv2019

Barley RIC157 is involved in RACB-mediated susceptibility to powdery mildew

Successful obligate pathogens benefit from host cellular processes. For the biotrophic ascomycete fungus Blumeria graminis f.sp. hordei (Bgh) it has been shown that barley RACB, a small monomeric G-protein (ROP, RHO of plants), is required for full susceptibility to fungal penetration. The susceptibility function of RACB probably lies in its role in cell polarisation, which may be co-opted by the pathogen for invasive ingrowth of its haustorium. However, the actual mechanism of how RACB supports the fungal penetration success is little understood. RIC proteins (ROP-Interactive and CRIB-(Cdc42/Rac Interactive Binding) motif-containing) are considered scaffold proteins which can interact directly with ROPs via a conserved CRIB motif. Here we describe a yet uncharacterised RIC protein, RIC157, which can interact directly with RACB in planta. We show that RIC157 undergoes a recruitment from the cytoplasm to the cell periphery in the presence of activated RACB. During fungal infection, RIC157 and activated RACB colocalise at the penetration site, particularly at the haustorial neck. In a RACB-dependent manner, transiently overexpressed RIC157 renders barley epidermal cells more susceptible to fungal penetration. This suggests that RIC157 promotes fungal penetration into barley epidermal cells via its function downstream of RACB.

plant biology↗

bioRxiv2019

ROP INTERACTIVE PARTNER b interacts with the ROP GTPase RACB and supports fungal penetration into barley epidermal cells

RHO of Plants (ROP) G-proteins are key components of cell polarization processes in plant development. The barley (Hordeum vulgare) ROP protein RACB, is a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). RACB also drives polar cell development, and this function might be coopted during formation of fungal haustoria in barley epidermal cells. In order to understand RACB signaling during the interaction of barley with Bgh, we searched for potential downstream interactors of RACB. Here, we show that ROP INTERACTIVE PARTNER b (RIPb, synonym: INTERACTOR OF CONSTITUTIVE ACTIVE ROP b; ICRb) directly interacts with RACB in yeast and in planta. Over-expression of RIPb supports susceptibility of barley to Bgh. RIPb further interacts with itself at microtubules. However, the interaction with activated RACB takes place at the plasma membrane. Both, RIPb and RACB are recruited to the site of fungal attack around the neck of developing haustoria suggesting locally enhanced ROP activity. We further assigned different functions to different domains of the RIPb protein. The N-terminal coiled-coil CC1 domain is required for microtubule localization, while the C-terminal coiled-coil CC2 domain is sufficient to interact with RACB and to fulfill a function in susceptibility at the plasma membrane. Hence, RIPb appears to be localized at microtubules and is then recruited by activated RACB for a function at the plasma membrane during formation of the haustorial complex. One Sentence summaryRIPb acts downstream of the powdery mildew susceptibility factor RACB of barley and influences susceptibility

plant biology↗