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Dendrites and dendritic spines are the essential cellular compartments in neuronal communication, conveying information through transient voltage signals. Our understanding of these compartmentalized voltage dynamics in fine, distal neuronal dendrites...

Funder: infinitus china ltd., In this review, we discuss the synaptic aspects of Tau pathology occurring during Alzheimer’s disease (AD) and how this may relate to memory impairment, a major hallmark of AD. Whilst the clinical diagnosis of AD patients is a loss of working memory and long-term declarative memory, the histological diagnosis is the presence of neurofibrillary tangles of hyperphosphorylated Tau and Amyloid-beta plaques. Tau pathology spreads through synaptically connected neurons to impair synaptic function preceding the formation of neurofibrillary tangles, synaptic loss, axonal retraction and cell death. Alongside synaptic pathology, recent data suggest that Tau has physiological roles in the pre- or post- synaptic compartments. Thus, we have seen a shift in the research focus from Tau as a microtubule-stabilising protein in axons, to Tau as a synaptic protein with roles in accelerating spine formation, dendritic elongation, and in synaptic plasticity coordinating memory pathways. We collate here the myriad of emerging interactions and physiological roles of synaptic Tau, and discuss the current evidence that synaptic Tau contributes to pathology in AD.

Graphene and graphene-related materials (GRMs) exhibit a unique combination of electronic, optical, and electrochemical properties, which make them ideally suitable for ultrasensitive and selective point-of-care testing (POCT) devices. POCT device-based applications in diagnostics require test results to be readily accessible anywhere to produce results within a short analysis timeframe. This review article provides a summary of methods and latest developments in the field of graphene and GRM-based biosensing in POCT and an overview of the main applications of the latter in nucleic acids and enzymatic biosensing, cell detection, and immunosensing. For each application, we discuss scientific and technological advances along with the remaining challenges, outlining future directions for widespread use of this technology in biomedical applications.

ZusammenfassungDie weltweit steigende Prävalenz von Übergewicht und Adipositas betrifft zunehmend Frauen im fertilen Alter. Ungewollte oder ungeplante Schwangerschaften treten bei Frauen mit Übergewicht häufiger auf, was sowohl auf die mangelnde Anwendung einer effektiven Kontrazeption als auch auf die pathophysiologischen Besonderheiten, die mit einem erhöhten Körperfettanteil einhergehen, zurückzuführen ist. Eine adäquate Kontrazeptionsberatung adipöser Frauen ist daher essenziell für ihre Gesundheit und Lebensqualität. Unter den kombinierten Kontrazeptiva stellen orale Präparate, die Ethinylestradiol und Levonorgestrel (LNG) enthalten, eine effiziente Option dar, vorausgesetzt, es bestehen zusätzlich zum erhöhten Body-Mass-Index (BMI) keine weiteren Risikofaktoren für kardiovaskuläre oder thrombembolische Ereignisse. Rein gestagene Präparate zeichnen sich durch ihre geringen gesundheitlichen Risiken aus. Die Angst vor einer weiteren Gewichtszunahme ist ein häufiger Grund für das Sistieren der Einnahme, wobei dieser Zusammenhang außer für Depot-Medoxyprogesteronacetat für die meisten Präparate wissenschaftlich nicht belegt wurde. Die Wirksamkeit intrauteriner Kontrazeptiva entfaltet sich unabhängig vom Körpergewicht. Als Notfallkontrazeption ist die Einlage eines kupferhaltigen Intrauterinpessars die sicherste Methode, jedoch erheblich aufwendiger und teurer als eine orale Notfallkontrazeption. Die orale Einnahme von Ulipristalacetat 30 mg ist aufgrund der potenteren Ovulationshemmung und der stärkeren Reduktion unerwünschter Schwangerschaften gegenüber LNG 1,5 mg zu bevorzugen.

Peer reviewed: True, Funder: Sorbonne Université, Funder: University of Cambridge, The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen’s health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device’s operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen’s cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen’s health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device’s operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen’s cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

SummaryAberrant condensation and localisation of the RNA-binding protein fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is also associated with cytoskeletal defects, genetically and through observations of compromised axonal transport. Here, we asked whether compromised axonal cytoskeletal organisation is an early feature of FUS-associated ALS/FTD. We used an ALS-associated mutant FUS(P525L) and the FTD-mimic hypomethylated FUS, FUS(16R), to investigate the common and distinct cytoskeletal changes found in these two reportedXenopusmodels. Combining a novel atomic force microscopy (AFM)-based approach forin vitrocytoskeletal characterisation andin vivoaxonal branching analysis, we found that mutant FUS reduced actin density in the dynamically remodelling growth cone, and reduced axonal branch complexity. We furthermore found evidence of an axon looping defect for FUS(P525L). Therefore, we show that compromised actin remodelling is potentially an important early event in FUS-associated pathogenesis.Abstract Figure

Monomeric alpha-synuclein (aSyn) is a well characterised as a lipid binding protein. aSyn is known to form amyloid fibrils which are also localised with lipids and organelles in so called Lewy bodies, insoluble structures found in Parkinson’s disease patient’s brains. It is still unclear under which conditions the aSyn-lipid interaction can start to become pathological. Previous work to address pathological interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes which not only have a more complex lipid composition, but also contain lipid interacting proteins. Here, we investigate how either monomeric or fibrillar aSyn interact with physiological synaptic vesicles (SV) isolated from rodent brain. Using small angle neutron scattering and high-resolution imaging we observe that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV. Furthermore, SV enhance the aggregation rate of aSyn, however increasing the SV:aSyn ratio causes a reduction in aggregation propensity. SV lipids appear as an integrated part of aSyn fibrils and while the fibril morphology differs to aSyn fibrils alone, the core fibril structure remains the same. We finally demonstrate that lipid-associated aSyn fibrils are more easily taken up into cortical i3Neurons derived from induced pluripotent stem cells. Our study sheds light on differences between interactions of aSyn with synthetic lipid vesicles and physiological SV. We show how aSyn fibrils may enhance pathology by disintegrating SV, which in turn may have fatal consequences for neurons. Furthermore, disease burden may additionally be impacted by an increased uptake of lipid-associated aSyn by neurons, leading to more SV damage and enhancing aSyn aggregation.

SummaryThe molecular mechanisms that connect the formation of aberrant cytoplasmic FUS condensates to biological malfunction are incompletely understood. Here, we develop an approach to determine the intracellular FUS viscosity in live mammalian cells and find that ALS-related mutant P525L-FUS forms the most viscous condensates and has impaired cytoskeletal mechanoproperties and increased euchromatin formation. We further show that some of the main cellular organelles, e.g., actin/tubulin, lysosomes, mitochondria, the endoplasmic reticulum, are significantly functionally/structurally impaired in the presence of FUS. These may be related to defects in the tubulin network, as the latter facilitates transport, formation, fusion and fission of organelles. We observe significant increases in lysosomal biogenesis, size and pH; moreover, intracellular FUS accumulation significantly promotes cytoplasmic-to-nuclear translocation of TFEB, i.e., the master gene for inducing autophagy. However, despite these, increased autophagy needed for protein aggregate clearance is not observed to occur. Our study reveals that the formation of highly viscous FUS condensates significantly impacts cytoskeletal/organelle function and cellular homeostasis, which are closely associated with cell ageing. This raises the intriguing question as to whether mutant FUS activates similar cell processes as those during cellular senescence.

Neurodegenerative disorders are a family of diseases that remain poorly treated despite their growing global health burden. A shared feature of many neurodegenerative disorders is the accumulation of toxic misfolded proteins. To gain insight into the mechanisms and modulators of protein misfolding, we developed a multiplex reverse genetics platform. Using this novel platform 29 cell-based models expressing proteins that undergo misfolding in neurodegeneration were probed against more than a thousand genetic modifiers. The resulting data provide insight into the nature of modifiers that act on multiple misfolded proteins as compared to those that show activity on only one. To illustrate the utility of this platform, we extensively characterized a potent hit from our screens, the human chaperone DNAJB6. We show that DNAJB6 is a general modifier of the toxicity and solubility of multiple amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD)-linked RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1. Biophysical examination of DNAJB6 demonstrated that it co-phase separates with, and alters the behavior of FUS containing condensates by locking them into a loose gel-like state which prevents their fibrilization. Domain mapping and a deep mutational scan of DNAJB6 support the critical importance for DNAJB6 phase separation in its effects on multiple RNA-binding proteins. Crucially, these studies also suggest that this property can be further tuned to generate novel variants with enhanced activity that might illuminate potential avenues for clinical translation.

The Alzheimer's disease related peptide, Amyloid-beta (Aβ)1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction, or the need to reconstitute Aβ from a freeze-dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The washing, solubilization, and purification steps take only 3 h. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

Funder: Infinitus China Ltd., Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.

Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson’s disease. A large body of evidence shows that α-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 Parkinson’s disease, that α-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 α-synuclein condensates in the presence of lipids and other cellular components.

Temperature is a fundamental physical parameter that influences biological processes in living cells. Hence, intracellular temperature mapping can be used to derive useful information reflective of thermodynamic properties and cellular behaviour. Herein, existing publications on different thermometry systems, focusing on those that employ fluorescence-based techniques, are reviewed. From developments based on fluorescent proteins and inorganic molecules to metal nanoclusters and fluorescent polymers, the general findings of intracellular measurements from different research groups are discussed. Furthermore, the contradiction of mitochondrial thermogenesis and nuclear-cytoplasmic temperature differences to current thermodynamic understanding are highlighted. Lastly, intracellular thermometry is proposed as a tool to quantify the energy flow and cost associated with amyloid-β42 (Aβ42) aggregation, a hallmark of Alzheimer's disease.

The topology of endoplasmic reticulum (ER) network is highly regulated by various cellular and environmental stimuli and affects major functions such as protein quality control and the cell’s response to metabolic changes. The ability to quantify the dynamical changes of the ER structures in response to cellular perturbations is crucial for the development of novel therapeutic approaches against ER associated diseases, such as hereditary spastic paraplegias and Niemann Pick Disease type C. However, the rapid movement and small spatial dimension of ER networks make this task challenging. Here, we combine video-rate super-resolution imaging with a state-of-the-art semantic segmentation method capable of automatically classifying sheet and tubular ER domains inside individual cells. Data are skeletonised and represented by connectivity graphs to enable the precise and efficient quantification and comparison of the network connectivity from different complex ER phenotypes. The method, called ERnet, is powered by a Vision Transformer architecture, and integrates multi-head self-attention and channel attention into the model for adaptive weighting of frames in the time domain. We validated the performance of ERnet by measuring different ER morphology changes in response to genetic or metabolic manipulations. Finally, as a means to test the applicability and versatility of ERnet, we showed that ERnet can be applied to images from different cell types and also taken from different imaging setups. Our method can be deployed in an automatic, high-throughput, and unbiased fashion to identify subtle changes in cellular phenotypes that can be used as potential diagnostics for propensity to ER mediated disease, for disease progression, and for response to therapy.

Inhibiting the aggregation of amyloid β (1-42) is a promising strategy for the development of disease-modifying Alzheimer’s disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic “nanoFLIM” assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid β self-assembly in vitro as well as in cellular and whole organism disease models.