Biology subjects

Schwarz, P.

Publications and source records attributed to Schwarz, P..

14 recordsLinked to original sources

bioRxiv2026

Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes

For decades gene amplifications were described as an attribute of tumor cells and as a physiological mechanism to increase gene copy numbers for the higher protein demand during development of amphibians and flies. An increasing number of publications describe gene amplifications in normal mammalian cells during differentiation. Many amplified genes detected in tumor cells overlap with amplified genes detected during stem cell differentiation. Since stem cells have a valuable potency in regenerative therapies and since cartilage regeneration is a highly demanded therapeutic strategy, we investigated gene amplification dynamics during chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Using quantitative PCR, we analyzed copy number changes for genes previously implicated in differentiation as well as genes amplified in chondrosarcoma including CDK4, MDM2, AGAP2, CPT1B, SHANK3, TRIB1, and MYC. Amplifications were transient and stage-specific: CDK4, CPT1B, and SHANK3 exhibited the highest copy number increases at day 2, followed by a gradual decline by day 7, while AGAP2 and MDM2 increased later in differentiation. Laser microdissection of toluidine blue-stained areas revealed heterogeneity in amplification patterns: CDK4 amplification was prominent in areas lacking or showing moderate proteoglycan deposition; CPT1B amplification occurred in regions with absent, moderate, or intense proteoglycan deposition; and SHANK3 amplification was restricted to areas with intense proteoglycan deposition. Notably, regions with the strongest proteoglycan staining exhibited no gene amplification, suggesting that gene amplification is an early, transient event that diminishes as differentiation progresses. These findings highlight gene amplification as a mechanism during chondrogenesis, potentially critical for early differentiation stages and genome stability in mature cells.

Developmental Biology↗

bioRxiv2024

Chemotherapy causes a reversible decrease in VMP1/MIR21 DNA methylation in granulocytes from breast cancer survivors

BackgroundDNA methylation alterations within the VMP1/MIR21 gene region, a potential epigenetic biomarker of systemic inflammation, have been demonstrated in mononuclear blood cells from early breast cancer (BC) patients after chemotherapy. Whether these changes are present in granulocytes, persist in the years after treatment, and affect VMP1 or MIR21 gene expression, remains unknown. AimWe aimed to investigate whether adjuvant chemotherapy alters the DNA methylation and gene expression of VMP1/MIR21 in granulocytes from postmenopausal BC patients and, if so, whether these treatment-induced changes are reversible in the first two years after completed chemotherapy. MethodsWhole blood samples were obtained from 30 postmenopausal BC patients before chemotherapy and every six months for two years, and from 10 healthy age- and BMI-matched controls. DNA and RNA was extracted from isolated granulocytes, and DNA methylation of four CpG sites located in the gene body of VMP1, which is situated in the promoter region of MIR21, was assessed through bisulfite pyrosequencing. qPCR was used for assessment of VMP1 and MIR21 expression. ResultsVMP1/MIR21 was significantly demethylated in granulocytes from BC patients shortly after completed chemotherapy compared to before (10 percentage points decrease, p<0.0001). Six months thereafter, DNA methylation values were significantly increased (6 percentage points, p = 0.002), and they were further increased to pre-chemotherapy levels 12, 18 and 24 months post chemotherapy. Chemotherapy did not cause significant changes in the expression of VMP1 or MIR21. ConclusionThe unique follow-up samples in this study demonstrated that chemotherapy induced a transient reduction in DNA methylation of the VMP1/MIR21 region in granulocytes from postmenopausal BC patients. Although transient, chemotherapy-induced epigenetic changes in blood cells may contribute to the increased risk of inflammatory-related comorbidities in BC survivors.

molecular biology↗

bioRxiv2024

DeepRod: A human-in-the-loop system for automatic rodent behavior analysis

We present a human-in-the-loop system for efficient rodent behavior analysis in drug development. Addressing the time-consuming and labor-intensive nature of manual behavior categorization, this UX-optimized platform integrates AI for complex behavior prediction and active learning to identify rare events. The proposed solution leverages a cloud-native data processing pipeline, AI-based novelty behavior recognition and multi-class classification, demonstrating significant improvements in behavior labeling and discovery.

animal behavior and cognition↗

bioRxiv2023

Skeletal-Muscle Glutamine Synthase is Upregulated in Preclinical Prion Diseases

In prion diseases, aggregates of misfolded prion protein (PrPSc) accumulate not only in the brain but also in extraneural organs. This raises the question whether prion-specific pathologies arise also extraneurally. Here we sequenced mRNA transcripts in skeletal muscle, spleen and blood of prion-inoculated mice at eight timepoints during disease progression. We detected gene-expression changes in all three organs, with skeletal muscle showing the most consistent alterations. The glutamate-ammonia ligase (GLUL) gene exhibited uniform upregulation in skeletal muscles of mice infected with three distinct scrapie prion strains (RML, ME7, and 22L) and in victims of human sporadic Creutzfeldt-Jakob disease. GLUL dysregulation was accompanied by changes in glutamate/glutamine metabolism, leading to reduced glutamate levels in skeletal muscle. None of these changes were observed in skeletal muscle of humans with amyotrophic lateral sclerosis, Alzheimers disease, or dementia with Lewy bodies, suggesting that they are specific to prion diseases. These findings reveal an unexpected metabolic dimension of prion infections and point to a potential role for GLUL dysregulation in the glutamate/glutamine metabolism in prion-affected skeletal muscle.

neuroscience↗

bioRxiv2023

A screen of alkaline and oxidative formulations for their inactivation efficacy of metal surface adsorbed prions using a steel-bead seed amplification assay

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the development of TESSA (sTainlESs steel-bead Seed Amplification assay), a prion seed amplification assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for the bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

neuroscience↗

bioRxiv2022

Peripheral blood mononuclear cells exhibit increased mitochondrial respiration after adjuvant chemo- and radiotherapy for early breast cancer

BackgroundAdjuvant chemo- and radiotherapy cause cellular damage not only to cancerous but also to healthy dividing cells. Antineoplastic treatments have been shown to cause mitochondrial respiratory dysfunction in non-tumorous tissues, but the effects on circulating human peripheral blood mononuclear cells (PBMCs) remain unknown. AimWe aimed to identify changes in mitochondrial respiration of PBMCs after adjuvant chemo- and radiotherapy in postmenopausal early breast cancer (EBC) patients and relate these to metabolic parameters of the patients. MethodsTwenty-three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy treatment often administered in combination with radiotherapy (n=18). Respiration (O2 flux per million PBMCs) was assessed by high-resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics were furthermore assessed. ResultsRespiration of intact and permeabilized PBMCs from EBC patients was significantly increased after adjuvant chemo- and radiotherapy (p=6x10-5 and p=1x10-7, respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17-43% compared to before therapy commencement. Leukocyte counts (p=1x10-4), hemoglobin levels (p=0.0003), and HDL cholesterol (p=0.003) decreased while triglyceride (p=0.01) and LDL levels (p=0.02) increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters correlated significantly with PBMC respiration. ConclusionThis study shows that mitochondrial respiration in circulating PBMCs is significantly increased after adjuvant chemo- and radiotherapy in postmenopausal EBC patients. The increase might be explained by a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation rather than glycolysis or a generally increased mitochondrial content in PBMCs. Both parameters might be influenced by therapy-induced changes to the bone marrow or vascular microenvironment wherein PBMCs differentiate and reside.

cell biology↗

bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗

bioRxiv2021

The ASC Protein Controls the Severity of Systemic AA Amyloidosis in Mice

ASC-containing inflammasomes form specks, extracellular aggregates which enhance the aggregation of A{beta} amyloid in Alzheimers disease. This raises the question whether ASC participates to additional aggregation proteinopathies. Here we show that ASC controls the extent of inflammation-associated AA amyloidosis, a systemic disease caused by the aggregation of the acute-phase reactant serum amyloid A (SAA). Using superresolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Purified recombinant ASC specks accelerated SAA fibril formation in vitro. Mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain. In a murine model of inflammation-associated AA amyloidosis, splenic AA amyloid load was conspicuously decreased in Pycardtm1Vmd/tm1Vmd mice which lack ASC. This reduction was not a consequence of enhanced amyloid phagocytosis, as SAA stimulation increased phagocytic activity in Pycard+/+, but not in Pycard-/- macrophages. Treatment with anti-ASC antibodies decreased the amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC50 [≥] 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. Higher anti-ASC titers did not correlate with any specific disease, suggesting that anti-ASC immunotherapy may be well-tolerated. These findings expand the role played by ASC to extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.

molecular biology↗

bioRxiv2021

Glial activation in prion diseases is strictly nonautonomous and requires neuronal PrPSc

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons, astrocytes or oligodendrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

neuroscience↗

bioRxiv2020

Prions induce minor genome-wide translational changes in neurons compared to glia

Prion diseases are caused by PrPSc, a self-replicating pathologically misfolded protein that exerts toxicity predominantly in the brain. The administration of PrPSc causes a robust, reproducible and specific disease manifestation. Here we have applied a combination of translating ribosome affinity purification and ribosome profiling to identify biologically relevant prion-induced changes during disease progression in a cell-type specific and genome-wide manner. Terminally diseased mice with severe neurological symptoms showed extensive alterations in astrocytes and microglia. Surprisingly, we detected only minor changes in the translational profiles of neurons. Prion-induced alterations in glia overlapped with those identified in other neurodegenerative diseases, suggesting that similar events occur in a broad spectrum of pathologies. Our results suggest that aberrant translation within glia may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.

neuroscience↗

bioRxiv2020

Synaptic accumulation of FUS triggers age-dependent misregulation of inhibitory synapses in ALS-FUS mice

FUS is a primarily nuclear RNA-binding protein with important roles in RNA processing and transport. FUS mutations disrupting its nuclear localization characterize a subset of amyotrophic lateral sclerosis (ALS-FUS) patients, through an unidentified pathological mechanism. FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Here, we used super-resolution imaging to determine the physiological localization of extranuclear, neuronal FUS and found it predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosome preparations, we identified synaptic RNA targets of FUS that are associated with synapse organization and plasticity. Synaptic FUS was significantly increased in a knock-in mouse model of ALS-FUS, at presymptomatic stages, accompanied by alterations in density and size of GABAergic synapses. RNA-seq of synaptoneurosomes highlighted age-dependent dysregulation of glutamatergic and GABAergic synapses. Our study indicates that FUS accumulation at the synapse in early stages of ALS-FUS results in synaptic impairment, potentially representing an initial trigger of neurodegeneration.

neuroscience↗

bioRxiv2020

Localization of hyphal growth associated with mycotoxin production during the malting of Fusarium head blight infected grains

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Objectives of the current study were to localize the growth of Fusarium within FHB infected kernels and to associate it with the production of DON that occurred during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in Fusarium Tri5 DNA and trichothecene mycotoxins following malting, were screened for hyphal localization. The growth of hyphae, both on the surface of kernels and within tissues of grain and malt was, imagined by scanning electron microscopy and confocal laser scanning microscopy assisted with WGA-Alexa Fluor 488 pre-staining, respectively. In barley, hyphae were primarily present on or within husk, vascular bundle, and pericarp cavities. Following malting, large amounts of hyphal growth were observed in not only these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. Interestingly, these grains already had an extensive internal presence of hyphae in unmalted grain, occurring in the pericarp, testa, vascular bundle, nucellar projection, aleurone layer, endosperm, pericarp and endosperm cavities, and embryo. Shotgun sequencing followed by metagenomics analysis verified that Fusarium spp. accounted for above 90% of the fungal hyphae growing in the interior of grains during malting, which coincided with the significant production of mycotoxins.

plant biology↗

bioRxiv2020

Genome-wide transcriptomics identifies an early preclinical signature of prion infection

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance, splicing and editing alterations during the course of disease in prion-inoculated mice. Prion infection induced transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical symptoms, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, represents the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed symptoms in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

neuroscience↗

bioRxiv2019

Transmission of prion infectivity from blood by peptoid-conjugated beads

Prions cause transmissible infectious diseases in humans and animals and have been found to be transmissible by blood transfusion even in the presymptomatic stage. However, the concentration of prions in body fluids such as blood and urine is extremely low, and therefore direct diagnostic tests on such specimens often yield false-negative results. Quantitative preanalytical prion enrichment may significantly improve the sensitivity of prion assays by concentrating trace amounts of prions from large volumes of body fluids. Here we show that beads conjugated to positively charged peptoids not only captured PrP aggregates from plasma of prion-infected hamsters, but also adsorbed prion infectivity in both the symptomatic and preclinical stages of the disease. Bead absorbed prion infectivity efficiently transmitted disease to transgenic indicator mice. We found that the readout of the peptoid-based misfolded protein assay (MPA) correlates closely with prion infectivity in vivo, thereby validating the MPA as a simple, quantitative, and sensitive surrogate indicator of the presence of prions. The reliable and sensitive detection of prions in plasma will enable a wide variety of applications in basic prion research and diagnostics.

neuroscience↗