Biology subjects

Sinnige, T.

Publications and source records attributed to Sinnige, T..

12 recordsLinked to original sources

bioRxiv2026

Polyglutamine aggregation proceeds by stochastic nucleation in the C. elegans nervous system

Fibrillar protein aggregation is the hallmark of a wide variety of neurodegenerative diseases. It is still poorly understood how soluble proteins transition to fibrillar aggregates, in particular within the complex environment of cells and tissues. Here, we employ the nematode Caenorhabditis elegans to elucidate the molecular mechanisms of polyglutamine aggregation in the nervous system of a multicellular animal. By using fluorescence microscopy in combination with mathematical modelling, we find that polyglutamine aggregation occurs by stochastic nucleation in individual neurons, at a rate that is of the same order of magnitude as previously determined for muscle cells. The J-protein DNAJB6 strongly suppresses polyglutamine aggregation in both tissues, consistent with a mechanism in which aggregation is limited by primary nucleation. We confirm that the same mechanism holds true for a construct including the disease-relevant flanking regions of Huntingtin exon 1. Altogether, our data reveal that polyglutamine aggregation occurs spontaneously and largely independent of the cellular context.

biophysics↗

bioRxiv2025

Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation

Tauopathies are a group of progressive neurodegenerative diseases amongst which Alzheimers disease is the most prevalent. They typically have a late age of onset and lead to cognitive decline. Tau is a highly soluble, intrinsically disordered microtubule binding protein that aggregates in these diseases. The mechanisms by which age-associated changes alter tau functionality and compromise neuronal structural integrity remain poorly understood. Here, we show with the use of single-copy gene insertion Caenorhabditis elegans models that human tau preferentially localises to neuronal processes in the nematode, consistent with its function of binding to microtubules. The expression of tau in the nervous system leads to age-associated changes in neuronal structural integrity. The observed "buckling" phenotype was exacerbated by exposure of the animals to different stressors which are known to enhance tau phosphorylation. Expressing phosphomimic tau or co-expressing human kinases both severely worsened the buckling phenotype. Kinase expression additionally induced tau inclusions in a small proportion of animals. These results point to an interplay between ageing, stress and phosphorylation leading to structural changes in neuronal processes prior to widespread tau aggregation.

neuroscience↗

bioRxiv2025

Urolithin B reduces the aggregate load of islet amyloid polypeptide in Caenorhabditis elegans

The progressive loss of pancreatic {beta}-cells is one of the defining features of Type 2 Diabetes Mellitus (T2DM), and is thought to be driven by the aggregation of islet amyloid polypeptide (IAPP). This highly amyloidogenic pancreatic hormone is co-secreted with insulin, and its elevated secretion can lead to toxic fibrillar aggregation. Despite numerous studies focusing on understanding the molecular mechanisms of IAPP aggregation, few therapeutic strategies exist to counter its toxicity. Urolithin B is a natural metabolite derived from the digestion and intestinal microbiota action on ellagitannin-rich foods. This compound was suggested to counteract IAPP aggregation and toxicity in silico and in yeast models expressing human IAPP. In this study, we focused on the protective potential of urolithin B using our previously characterised transgenic Caenorhabditis elegans IAPP-GFP model. We report that urolithin B reduces the levels of insoluble IAPP-GFP in the body wall muscle cells, while the mitochondrial association of IAPP-GFP remains unaltered. We show that the C. elegans model exhibits a reduced lifespan compared to controls, providing in vivo evidence of the toxic effects associated with IAPP-GFP expression. The lifespans of IAPP-GFP versus control animals were differentially modulated by urolithin B treatment, suggesting an interplay between the metabolite and IAPP-GFP mediated toxicity. To further elucidate the mode of action of urolithin B, we conducted in vitro assays and found no evidence of direct interaction with lipid-associated IAPP, suggesting that its effects are not mediated by interference with IAPP-membrane interactions. These results pave the way for further therapeutic developments targeting IAPP aggregation in T2DM.

biochemistry↗

bioRxiv2024

Novel hepatocyte-like liver organoids recapitulate crucial mature hepatic functions

Accurate liver disease modeling and drug toxicity testing still remain challenging as liver cells in vitro poorly resemble adult hepatocytes, as we previously demonstrated using whole transcriptome and cell identity analysis. To address this, we used our insights into hepatic modeling to develop hepatocyte-like liver organoids (HeLLOs), a novel human organoid model with mature hepatocyte functions superior to existing models. HeLLOs are easily established from (small) healthy or diseased liver tissues and rapidly expanded for an extended period in optimized culture conditions. Transcriptomic and functional analyses revealed that differentiated HeLLOs closely resemble fresh primary human hepatocytes (PHHs) and perform key hepatic functions such as gluconeogenesis, drug metabolism, and bile acid synthesis. We developed a HeLLO-based toxicity assay with higher sensitivity in predicting liver toxicity of known liver-toxic drugs compared to the gold-standard PHHs. By modeling disease-related mechanisms, such as bile acid transport, HeLLOs uncover transport-inhibition toxicity mechanisms of known liver toxic drugs. Single cell sequencing analysis of HeLLOs identified a heterogeneous cluster of cells with cholangiocyte-like and hepatocyte-like cells, overall resembling liver regenerative cells. As such, HeLLOs hold great promise for advancing liver disease modeling and drug testing. To our knowledge, HeLLOs are the best expandable liver model for predicting adverse drug reactions as well as modeling various liver disease mechanisms.

cell biology↗

bioRxiv2024

A hollow fiber membrane-based liver organoid-on-a-chip model for examining drug metabolism and transport

Liver-on-a-chip models predictive for both metabolism as well as canalicular and blood transport of drug candidates in humans are lacking. Here, we established an advanced, bioengineered and animal component-free hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g., CYP3A4, CYP2D6) and II (UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g., OATP1B3, MDR1 and MRP3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we connected the system to the other two PK/ADME-most relevant organ systems, i.e. small intestine- and kidney proximal tubule-like to study the bioavailability of midazolam and coumarin, and excretion of metformin. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict PK/ADME profiles in humans.

pharmacology and toxicology↗

bioRxiv2023

Islet amyloid polypeptide tagged with green fluorescent protein localises to mitochondria and forms filamentous aggregates in Caenorhabditis elegans

Type 2 diabetes (T2D) is the most common form of diabetes and represents a growing health concern. A characteristic feature of T2D is the aggregation of islet amyloid polypeptide (IAPP), which is thought to be associated with the death of pancreatic {beta}-cells. Inhibiting IAPP aggregation is a promising therapeutic avenue to treat T2D, but the mechanisms of aggregation and toxicity are not yet fully understood. Caenorhabditis elegans is a well-characterised multicellular model organism that has been extensively used to study protein aggregation diseases. In this study, we aimed to develop a simple in vivo model to investigate IAPP aggregation and toxicity based on expression in the C. elegans body wall muscle cells. We show that IAPP tagged with green fluorescent protein (GFP) localises to mitochondria in this tissue, in line with previous observations in mouse and human pancreatic {beta}-cells. The IAPP-GFP fusion protein forms solid aggregates, which have a filamentous appearance as seen by electron microscopy. However, the animals do not display a strong motility phenotype, suggesting that the IAPP-GFP aggregates are not considerably toxic. Nevertheless, the mitochondrial localisation and aggregate formation may be useful read-outs to screen for IAPP-solubilizing compounds as a therapeutic strategy for T2D. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/564377v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@98780aorg.highwire.dtl.DTLVardef@1f11767org.highwire.dtl.DTLVardef@13783c7org.highwire.dtl.DTLVardef@1acbe0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic {beta}-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage.

biochemistry↗

bioRxiv2022

Membrane-catalyzed IAPP aggregation is dominated by secondary nucleation

Type II diabetes is characterized by the loss of pancreatic {beta}-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, i.e. the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent {beta}-cell death in type II diabetes.

biophysics↗

bioRxiv2020

Kinetic analysis reveals the rates and mechanisms of protein aggregation in a multicellular organism

The accumulation of insoluble protein aggregates containing amyloid fibrils has been observed in many different human protein misfolding diseases1,2, and their pathological features have been recapitulated in diverse model systems3. In vitro kinetic studies have provided a quantitative understanding of how the fundamental molecular level processes of nucleation and growth lead to amyloid formation4. However, it is not yet clear to what extent these basic biophysical processes translate to amyloid formation in vivo, given the complexity of the cellular and organismal environment. Here we show that the aggregation of a fluorescently tagged polyglutamine (polyQ) protein into {micro}m-sized inclusions in the muscle tissue of living C. elegans can be quantitatively described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results are consistent with observations for the aggregation of polyQ proteins in vitro5 and in cell culture6, and highlight how nucleation events control the overall progression of aggregation in the organism through the spatial confinement into individual cells. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

biophysics↗

bioRxiv2020

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Misfolded -synuclein is a major component of Lewy bodies, which are a hallmark of Parkinsons disease. A large body of evidence shows that -synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinsons disease, that -synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of -synuclein condensates in the presence of lipids and other cellular components.

molecular biology↗

bioRxiv2018

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis and increased protein instability is a common feature of aging. Yet it remains unclear whether protein instability is a cause of aging. In neurodegenerative diseases and amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological solid aggregates in a variety of tissues. More recently, widespread protein aggregation has been described during normal aging, in the absence of disease processes. Until now, an extensive characterization of the nature of age-dependent protein aggregation and its consequences for aging has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and contain amyloid-like structures similar to disease-associated protein aggregates. Moreover, we demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these finding reveal that the formation of amyloid aggregates is a generic problem of aging and likely to be an important target for strategies designed to maintain physiological functions in later stages of life.

cell biology↗

bioRxiv2018

Fast fluorescence lifetime imaging reveals the maturation process of α-synuclein aggregates in ageing Caenorhabditis elegans

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinsons and Huntingtons diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by -synuclein and polyglutamine. In particular, we found that -synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

biophysics↗