Biology subjects

Wurst, W.

Publications and source records attributed to Wurst, W..

15 recordsLinked to original sources

bioRxiv2026

Ubiquitin ligase RNF126 exerts functions in lysosomal damage response and lysosomal homeostasis maintenance

The response to lysosomal membrane permeabilization involves extensive ubiquitylation at the lysosome; however, the ubiquitin ligases identified so far cannot account for the complexity of the response. Using extensive spatial proteomics, we identified RNF126 as a new ligase that is robustly recruited to lysosomes in response to a range of different damage triggers across distinct cell types. Recruitment is mediated independent of BAG6 via an arginine-rich stretch in the low-complexity region of RNF126 to mostly unacidified lysosomes in the perinuclear region. Ubiquitin remnant mass spectrometry identified several damage-induced RNF126 substrates, among them known lysosomal damage response components and potential new factors that might drive regeneration from lysosomal damage. Consistently, RNF126 deletion resulted in persistent lysosomal swelling. In addition, we uncovered a constitutive role of RNF126 in lysosomal homeostasis maintenance. Hence, our findings identified the ubiquitin ligase RNF126 as novel lysosomal health regulating factor with functions in homeostasis and stress conditions.

cell biology↗

bioRxiv2026

Novel mouse reporter models for the detection of genome editing events in vivo

With the expansion of therapeutic gene editing technology, small animal models provide essential platforms to evaluate the function of these new approaches in vivo. As part of the Somatic Cell Genome Editing (SCGE) Consortium, we developed next-generation murine reporters that overcome current model limitations and broaden detectable in vivo editing outcomes. These include two mouse models built on the "traffic light" reporter concept. This system enables fluorescent detection of both gene repair (green) and CRISPR-generated indels (red) events following editing by a single guide and either dsDNA or single-stranded oligonucleotide donor. We also generated a third reporter model that efficiently detects A-base editor activity. Reporters were validated in cultured embryos, via germline editing, and through activation in vivo by AAV transduction or direct ribonucleoprotein delivery. Together, these new models provide a valuable resource for improved detection of genome editing events in vivo.

genomics↗

bioRxiv2025

GluTrooper: a novel reporter mouse line for whole-brain imaging of glutamate dynamics

Glutamate is the primary excitatory neurotransmitter in the mammalian brain. However, tools to image glutamate dynamics in the whole brain with high spatial and temporal resolution are lacking. Therefore, we developed GluTrooper, a novel mouse line engineered for inducible and long-lasting expression of the genetically encoded glutamate sensor iGluSnFR3. GluTrooper mice crossed with Emx1-Cre lines demonstrated uniform and stable sensor expression in excitatory neurons of the cortex, hippocampus, and olfactory bulb. iGluSnFR3 expression remained stable for at least 12 months, enabling longitudinal observations of glutamate dynamics over extended periods. Using multimodal imaging in awake mice, we demonstrated the versatility of GluTrooper across multiple spatial scales: from mesoscale widefield cortical imaging to cellular resolution with two-photon microscopy. Moreover, during cortical spreading depolarization, bilateral whole-brain glutamate dynamics and contralateral cortical disinhibition were detected with high fidelity. Accordingly, the GluTrooper may open new avenues for the better understanding of glutamatergic neurotransmission in the mammalian brain.

neuroscience↗

bioRxiv2025

VMAT2 dysfunction impairs vesicular dopamine uptake, driving its oxidation and α-synuclein pathology in DJ-1-linked Parkinson's disease neurons

Parkinsons disease (PD) is characterized by -synuclein accumulation and dopaminergic neuron degeneration, with dopamine (DA) oxidation emerging as a key pathological driver. However, the mechanisms underlying this neurotoxic process remain unclear. Using PD patient-derived and CRISPR-engineered iPSC midbrain dopaminergic neurons lacking DJ-1, we identified defective sequestration of cytosolic DA into synaptic vesicles, which culminated in DA oxidation and -synuclein accumulation. In-depth proteomics, state-of-the-art imaging, and ultrasensitive DA probes uncovered that decreased VMAT2 protein and function impaired vesicular DA uptake, resulting in reduced vesicle availability and abnormal vesicle morphology. Furthermore, VMAT2 activity and vesicle endocytosis are processes dependent on ATP, which is notably reduced in DJ-1-deficient dopaminergic neurons. ATP supplementation restored vesicular function and alleviated DA-related pathologies in mutant dopaminergic neurons. This study reveals an ATP-sensitive mechanism that regulates DA homeostasis through VMAT2 and vesicle dynamics in midbrain dopaminergic neurons, highlighting enhanced DA sequestration as a promising therapeutic strategy for PD. TeaserLoss of DJ-1 interferes with VMAT2 function and vesicle dynamics, leading to DA oxidation and -synuclein pathology in PD neurons.

neuroscience↗

bioRxiv2024

TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and sequence variants are major risk factors for late onset Alzheimers disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling, we generated a TREM2 reporter mouse model and observed a gradual upregulation of reporter expression with increasing plaque proximity. Isolated microglia were sorted based on reporter expression and their transcriptomic profiles acquired in both wildtype and APP transgenic animals, allowing us to disentangle TREM2 versus pathology-specific effects. Bulk RNA-sequencing highlighted TREM2 level-dependent changes in major immunometabolic pathways, with enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. To confirm these findings, we next analysed uptake of fluorodeoxyglucose (FDG) and examined metabolomic and lipidomic profiles. Again, independent of A{beta} pathology, TREM2 expression correlated with uptake of FDG as well as increased cellular redox, energetics, and cholesterol homeostasis. Finally, we performed chronic treatment with a brain penetrant TREM2 agonist and identified a window of TREM2 expression where microglia are most responsive. Thus, our data provide novel insights into TREM2-mediated regulation of microglial metabolic function and informs current efforts to bring TREM2 agonists into clinical application.

neuroscience↗

bioRxiv2023

Comparative phenotyping of mice reveals canonical and noncanonical physiological functions of TRα and TRβ

Thyroid hormone (TH) effects are mediated through TH receptors (TRs) TR1, TR{beta}1, and TR{beta}2. The TRs bind to thyroid hormone responsive elements on the DNA and regulate expression of TH target genes as ligand dependent transcription factors (canonical signaling). In addition, the TRs and {beta} mediate activation of signaling pathways, e.g. the PI3K/AKT and MAPK/ERK pathways (noncanonical signaling). Whether such DNA-binding independent TR action contributes to the spectrum of physiological TH effects is largely unknown. The aim of this study was to attribute physiological effects to the two TR isoforms and {beta} and their canonical and noncanonical signaling. We conducted multi-parameter phenotyping in male and female TR knockout mice (TRKO, TR{beta}KO), mice with disrupted canonical signaling due to a mutation in the TR DNA-binding domain (TRGS, TR{beta}GS) and their respective wild-type littermates. Perturbations in senses, especially hearing (mainly TR{beta} with a lesser impact of TR), visual acuity and retinal thickness (TR and TR{beta}), in muscle metabolism (TR) and in heart rate (TR) highlighted the role of canonical TR action. Strikingly, selective abrogation of canonical TR action often had little to no phenotypic consequence, suggesting that noncanonical TR action sufficed to maintain the wild-type phenotype for specific effects. For instance, macrocytic anemia, reduced retinal vascularization or increased anxiety related behavior were only observed in TRKO, but not TRGS mice. Noncanonical TR action increased the efficiency of energy utilization and prevented hyperphagia observed in TRKO mice. In summary, by examining the phenotypes of TR and TR{beta} knockout models alongside their DNA-binding-deficient GS mutants and wildtype counterparts, we could establish that the independent noncanonical actions of TR and TR{beta} play a crucial role in modulating sensory, behavioral, and metabolic functions. This comparison underscores the significance of the TRs in orchestrating a spectrum of physiological processes beyond their traditional genomic pathways.

physiology↗

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↗

bioRxiv2022

Toxicity of extracellular alpha-synuclein is independent of intracellular alpha-synuclein

Parkinson's disease (PD) pathology progresses throughout the nervous system affecting numerous neuronal structures. It has been postulated that the progression of the pathology is based on a prion-like disease mechanism partly due to the seeding effect of endocytosed alpha-synuclein (ASYN) on the endogenous ASYN. The appearance of the pathology in dopaminergic neurons leads to neuronal cell death and motor symptoms. However, the effect on other neuronal structures is more inconsistent, leading to a higher variability in the prevalence of non-motor symptoms. Thus, the sensitivity to the pathology seems to vary among neuronal subtypes. Here, we analyzed the role of endogenous ASYN in the progression of PD-like pathology and the effect of monomeric and oligomeric ASYN as well as paraquat and rotenone on primary enteric, dopaminergic and cortical neurons from wild-type mice. Our results showed that pathology progression did not occur in the absence of endogenous ASYN and that dopaminergic neurons were more sensitive to ASYN and rotenone when compared to all other neuronal subtypes. Remarkably, the toxic effect of ASYN was independent of the presence of endogenous ASYN and directly related to the disturbance of the mitochondrial membrane potential. Thus, we suggest that the interaction between ASYN and mitochondria plays an important role in the toxicity of trans-synaptically transported ASYN and in the progression of PD pathology. These results question the prion-disease hypothesis and propose that endocytosed ASYN impairs the host's mitochondrial function thereby also contributing to PD-pathology progression.

neuroscience↗

bioRxiv2021

Susceptibility to diet induced obesity at thermoneutral conditions is independent of UCP1

ObjectiveActivation of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) upon cold stimulation leads to substantial increase in energy expenditure to defend body temperature. Increases in energy expenditure after a high caloric food intake, termed diet-induced thermogenesis, are also attributed to BAT. These properties render BAT a potential target to combat diet-induced obesity. However, studies investigating the role of UCP1 to protect against diet-induced obesity are controversial and rely on the phenotyping of a single constitutive UCP1-knockout model. To address this issue, we generated a novel UCP1-knockout model by Cre-mediated deletion of Exon 2 in the UCP1 gene. We studied the effect of constitutive UCP1 knockout on metabolism and the development of diet-induced obesity. MethodsUCP1 knockout and wildtype mice were housed at 30{degrees}C and fed a control diet for 4-weeks followed by 8-weeks of high-fat diet. Body weight and food intake were monitored continuously over the course of the study and indirect calorimetry was used to determine energy expenditure during both feeding periods. ResultsBased on Western blot analysis, thermal imaging and noradrenaline test, we confirmed the lack of functional UCP1 in knockout mice. However, body weight gain, food intake and energy expenditure were not affected by deletion of UCP1 gene function during both feeding periods. ConclusionConclusively, we show that UCP1 does not protect against diet-induced obesity at thermoneutrality. Further we introduce a novel UCP1-KO mouse enabling the generation of conditional UCP1-knockout mice to scrutinize the contribution of UCP1 to energy metabolism in different cell types or life stages.

physiology↗

bioRxiv2020

The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes beneficial microglial functions

BackgroundMicroglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimers disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) selectively expressed in microglia and macrophages was recently identified and shown to reduce the risk for AD. MethodsTo assess the role of this variant in the context of immune cell functions, we generated a Plc{gamma}2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. ResultsFunctional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plc{gamma}2 as a Pip2-metabolizing enzyme. This was associated with improved survival, enhanced phagocytic activity, and increased acute inflammatory response of the KI cells. Enhanced phagocytosis was also observed in mouse BV2 microglia-like cells overexpressing human PLC{gamma}2-P522R, but not in PLC{gamma}2-WT expressing cells. Furthermore, the brain mRNA signature together with microglia-specific PET imaging indicated microglia activation in Plc{gamma}2-P522R KI mice. ConclusionThus, we have delineated cellular mechanisms of the protective Plc{gamma}2-P522R variant, which provide further support for the emerging idea that activated microglia exert protective functions in AD.

neuroscience↗

bioRxiv2020

Canonical Wnt-signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons

In adult hippocampal neurogenesis neural stem/progenitor cells generate new dentate granule neurons that contribute to hippocampal plasticity. The establishment of a morphologically defined dendritic arbor is central to the functional integration of adult-born neurons. Here, we investigated the role of canonical Wnt/{beta}-catenin-signaling in dendritogenesis of adult-born neurons. We show that canonical Wnt-signaling follows a biphasic pattern, with high activity in stem/progenitor cells, attenuation in early immature neurons, and re-activation during maturation, and demonstrate that the biphasic activity pattern is required for proper dendrite development. Increasing {beta}-catenin-signaling in maturing neurons of young adult mice transiently accelerated dendritic growth, but eventually resulted in dendritic defects and excessive spine numbers. In middle-aged mice, in which protracted dendrite and spine development was paralleled by lower canonical Wnt-signaling activity, enhancement of {beta}-catenin-signaling restored dendritic growth and spine formation to levels observed in young adult animals. Our data indicate that precise timing and strength of {beta}-catenin-signaling is essential for the correct functional integration of adult-born neurons and suggest Wnt/{beta}-catenin-signaling as a pathway to ameliorate deficits in adult neurogenesis during aging.

neuroscience↗

bioRxiv2019

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinsons disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.

biochemistry↗

bioRxiv2019

PINK1 Regulates Dopamine and Lipids at Mitochondria to Maintain Synapses and Neuronal Function

Mitochondrial dysfunction contributes to the pathogenesis of Parkinsons disease but it is not clear why inherent mitochondrial defects lead specifically to the death of dopaminergic neurons of the mid brain. PINK1 is mitochondrial kinase and PINK1 mutations cause early onset Parkinsons disease.\n\nWe found that in neuronal progenitors, PINK1 regulates mitochondrial morphology, mitochondrial contact to the endoplasmic reticulum (ER) and the phosphorylation of Miro1. A compensatory metabolic shift towards lipid synthesis provides mitochondria with the components needed for membrane renewal and oxidative phosphorylation, maintaining the mitochondrial network once mature.\n\nCholesterol is increased by loss of PINK1, promoting overall membrane rigidity. This alters the distribution of phosphorylated DAT at synapses and impairs dopamine uptake. PINK1 is required for the phosphorylation of tyrosine hydroxylase at Ser19, dopamine and calcium homeostasis and dopaminergic pacemaking.\n\nWe suggest a novel mechanism for PINK1 pathogenicity in Parkinsons disease in addition to but not exclusive of mitophagy. We also provide a basis for potential therapeutics by showing that low doses of the cholesterol depleting drug {beta}-cyclodextrin reverse PINK1-specific phenotypes.

neuroscience↗

bioRxiv2019

Human and mouse essentiality screens as a resource for disease gene discovery

Although genomic sequencing has been transformative in the study of rare genetic diseases, identifying causal variants remains a considerable challenge that can be addressed in part by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from the comprehensive viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and from human cell line essentiality screens. We propose a novel, cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing characteristics in the biological processes they regulate, tissue expression levels and human mutation rates. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented in the developmental lethal category, representing genes not essential for cell survival but required for organism development. Exploiting this finding, we have screened developmental disorder cases from three independent disease sequencing consortia and identified potentially pathogenic, de novo variants shared in different patients for several developmental lethal genes that have not previously been associated with rare disease. We therefore propose FUSIL as an efficient resource for disease gene discovery.

genomics↗

bioRxiv2018

Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

Mitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

cell biology↗