Biology subjects

Knott, G.

Publications and source records attributed to Knott, G..

11 recordsLinked to original sources

bioRxiv2025

Recapitulating Parkinson's pathology in human iPSC dopaminergic neurons reveals new mechanistic insights into Lewy body formation and heterogeneity.

The accumulation of alpha-synuclein (aSyn) into intraneuronal inclusions of heterogeneous morphology, known as Lewy bodies (LB), is one of the defining diagnostic features of Parkinsons disease (PD); yet, our understanding of the mechanisms underpinning their formation and heterogeneity remains incomplete. Here, we present a human isogenic iPSC-derived dopaminergic neuron (iDA) model that faithfully recapitulates the diverse biochemical, morphological, and ultrastructural features of LB neuropathology in PD. Unlike other iDA seeding models, our model does not rely on aSyn overexpression, mutations, or genetic engineering, making it a more physiologically relevant system for studying PD. We demonstrate that the iDA model accurately reproduces the temporal relationships between neuritic and cell-body aSyn pathology, recapitulating the full biochemical spectrum, post-translational modifications (PTM), and morphological diversity of aSyn aggregates found in human PD tissue. Moreover, our work provides critical insight into how different pathways to aSyn fibrillization and the complex interaction between aSyn fibrils and membranous organelles influence the morphological diversity of LB-like inclusions. This model represents a versatile platform for investigating the mechanisms of pathology formation, maturation, and neuronal dysfunction, as well as supporting the development of diagnostics that capture the diversity of aSyn pathology in PD and related synucleinopathies.

neuroscience↗

bioRxiv2024

Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

Alpha-synuclein (aSyn) post-translational modifications (PTMs), particularly phosphorylation at serine 129 and C-terminal truncations, are highly enriched in Lewy bodies (LBs), Lewy neurites, and other types of aSyn pathological aggregates in the brain of patients with Parkinsons disease (PD) and other synucleinopathies. However, our knowledge about the precise role of PTMs in regulating the different stages of pathology formation, neurodegeneration, and aSyn pathology spreading remains incomplete. In this work, we applied a systematic approach to address this knowledge gap with an emphasis on mapping and elucidating the role of post-fibrillization C-terminal aSyn truncations in regulating the uptake, processing, seeding activity, and formation of LB-like inclusions and maturations in a well-established neuronal seeding model that recapitulates all the stages leading to LB formation and neurodegeneration. Our work shows that C-terminal cleavage of aSyn fibrils at multiple sites is a conserved process that occurs rapidly after and during the formation of intracellular LB-like aSyn inclusions in all neuronal seeding models. Interestingly, blocking the cleavage of internalized fibrils does not influence their seeding activity, whereas inhibiting the enzymes that regulate the cleavage of newly formed fibrils (e.g., calpains 1 and 2) significantly alters the formation of LB-like inclusions. We also show that C-terminal truncations, in combination with other PTMs, play a crucial role in regulating the interactome and remodeling of newly formed aSyn fibrils, including their shortening, lateral association, and packing during LB formation and maturation. Altogether, our results demonstrate that post-fibrillization C-terminal truncations have a differential role at different stages of aSyn aggregation and pathology formation. These insights, combined with the abundance of truncated aSyn species in LBs, have significant implications in understanding aSyn pathological diversity and developing therapeutic strategies targeting the C-terminus of aSyn or its proteolytic processing.

neuroscience↗

bioRxiv2024

Lamellar Schwann cells in the Pacinian corpuscle potentiate vibration perception

Pacinian corpuscles are among the most sensitive mechanoreceptors found in vertebrates and they are tuned to vibrations in the highest perceptible frequency range (100-2000Hz). One of their anatomical hallmarks is the onion-like cell layers surrounding the central axon. The innermost layers consist of [~]60 densely packed lamellar Schwann cells (LSCs), whose function remains largely unknown. Using high-resolution 3D electron microscopy we found that LSCs in Pacinian corpuscles of the mouse hindlimb do not form concentric rings, but complex, multilayered and intertwining assemblies that are connected via an estimated 5805.1 desmosomes and 4142.5 gap-junctions. LSCs make multiple converging contacts with the afferent axon and its protrusions with desmosomes. Using optogenetic manipulations of LSCs we demonstrate that their activation does not only drive reliable time-locked spiking in the axon, but that their inactivation significantly elevates the thresholds in-situ and increases perceptual thresholds behaviorally. Together these findings provide evidence that LSCs are a key element of somatosensory processing, actively potentiating mechanosensitivity in Pacinian corpuscles. HighlightsO_LIHigh-resolution electron microscopy reveals details of the Pacinian corpuscle C_LIO_LILamellar Schwann cells form claw-like structures with converging axonal contacts C_LIO_LISchwann-cell modulation bidirectionally affects neural coding of Pacinian afferent C_LIO_LIInactivation of lamellar Schwann-cells increases perceptual thresholds C_LI

neuroscience↗

bioRxiv2024

Obtaining high-resolution cryo-EM structures using a common LaB6, 120-keV electron microscope equipped with a sub 200-keV optimised direct electron detector.

Cryo-electron microscopy (Cryo-EM) single particle analysis (SPA) has become a major structural biology technique in recent years. High-resolution cryo-EM typically requires higher voltage cryo-TEMs with coherent FEG sources, stable columns, autoloader systems and direct electron detectors. These setups are specialised for Cryo-EM work and are expensive to establish and maintain. More recently the concept of using 100-keV cryo-TEMs has been introduced as a way to make cryo-EM more affordable and hence accessible to a larger group of researchers. So far, the implementation of these 100-keV cryo-TEMs have relied on specialised microscopes with FEG sources as well as more stable optics than usually present on the common 120-keV TEMs. We here explored whether a standard 120-keV TEM, commonly available at many laboratories worldwide, can be upgraded with a direct electron detector and its suitability for high-resolution cryo-EM using a standard side entry cryo-holder. Using this imaging configuration, we were successful in achieving a 2.65[A] reconstruction for standard apoferritin. We were also able to resolve a more challenging small 64kDa protein haemoglobin to 4.33[A]. Furthermore, we were able to solve an asymmetric 153 kDa membrane protein GPCR (M4 muscarinic acid receptor) to a resolution of 4.4[A]. Importantly, all these results were achieved using a standard automated data collection routine implemented through SerialEM, making it feasible to collect large cryo-EM data sets with a side entry cryo-holder. These results showcase a potentially widely accessible solution to obtaining interpretable cryo-EM structures. Furthermore, we envisage that this imaging configuration gives an option for many EM facilities and laboratories to set up a high-quality cryo-EM SPA sample screening capability without the need to procure costly specialised Cryo-TEMs. This could help to considerably lower the economic entry barrier for cryo-EM SPA and contribute to the "democratisation" of cryo-EM.

biophysics↗

bioRxiv2024

Mechanisms of axoneme and centriole elimination in Naegleria gruberi

The excavate Naegleria gruberi, a basal eukaryote related to the "brain eating" Naegleria fowleri, can transform transiently from an amoeboid life form lacking flagella and centrioles to a flagellate life form where these elements are present, followed by reversion to an amoeboid state. The mechanisms imparting elimination of axonemes and centrioles during this reversion process are not known. Here, we uncover that flagella primarily fold onto the cell surface and fuse within milliseconds with the plasma membrane. Once internalized, axonemes are severed by Spastin into equally-sized fragments, which are then enclosed by membranes, after which their contents are eliminated through the lysosomal pathway. Moreover, we discovered that centrioles undergo progressive K63 autophagy-linked poly-ubiquitination and K48 proteasome-promoting poly-ubiquitination, and that ubiquitination occurs next to centriolar microtubules. Most centrioles are eliminated in lysosomes or the cytoplasm in a lysosomal- and proteasome-dependent manner. Strikingly, we uncover in addition that centrioles can be shed in the extracellular milieu and taken up by other cells. Collectively, these findings reveal fundamental mechanisms governing the elimination of essential cellular constituents in Naegleria that may operate broadly in eukaryotic systems. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

cell biology↗

bioRxiv2023

A sexually dimorphic hepatic cycle of very low density lipoprotein uptake and assembly

Recent single-cell transcriptomes revealed spatiotemporal programmes of liver function on the sublobular scale. However, how sexual dimorphism affected this space-time logic remained poorly understood. We addressed this by performing scRNA-seq in the mouse liver, which revealed that sex, space and time together markedly influence xenobiotic detoxification and lipoprotein metabolism. The very low density lipoprotein receptor (VLDLR) exhibits a pericentral expression pattern, with significantly higher mRNA and protein levels in female mice. Conversely, VLDL assembly is periportally biased, suggesting a sexually dimorphic hepatic cycle of periportal formation and pericentral uptake of VLDL. In humans, VLDLR expression is also pericentral, with higher mRNA and protein levels in premenopausal women compared to similarly aged men. Individuals with low hepatic VLDLR expression show a high prevalence of atherosis in the coronary artery already at an early age and an increased incidence of heart attack.

physiology↗

bioRxiv2020

Invasion into the bladder wall generates solitary subpopulations of uropathogenic Escherichia coli that are protected from killing by antibiotics and neutrophil swarms in an organoid model

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) requiring antibiotic therapy. Recurrent infections, which occur in a quarter of treated individuals, may arise from "quiescent intracellular reservoirs" of bacteria that invade deeper layers of the bladder wall following infection and exfoliation of superficial umbrella cells. Here, we present a novel bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a small volume suitable for live-cell imaging of host-pathogen dynamics with high spatiotemporal resolution. We confirm that bacteria injected into the organoid lumen rapidly enter superficial cells that resemble umbrella cells and proliferate to generate tightly packed colonies that resemble intracellular bacterial communities (IBCs), a hallmark of UPEC pathogenesis. Unexpectedly, at early stages of infection we detect individual "solitary" bacteria that penetrate deeper layers of the organoid wall, where they evade killing by antibiotics and neutrophils. Volumetric serial block face scanning electron microscopy of infected organoids reveals that solitary bacteria can be found throughout the bladder wall and may be intracellular or pericellular (sandwiched between uroepithelial cells). Unlike bacteria within IBCs, which are coccoid-shaped and non-flagellated, solitary bacteria within the bladder wall are rod-shaped and flagellated. We conclude that early invasion of deeper layers of the bladder wall, independent of IBC formation, results in the establishment of reservoirs of solitary bacteria that resist elimination by antibiotics and the host innate immune response.

microbiology↗

bioRxiv2019

The process of Lewy body formation, rather than simply alpha-synuclein fibrillization, is the major driver of neurodegeneration in synucleinopathies

Parkinsons disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (-syn) into intraneuronal inclusions named Lewy bodies (LB). Although it is widely believed that -syn plays a central role in the pathogenesis of PD and synucleinopathies, the processes that govern -syn fibrillization and LB formation in the brain remain poorly understood. In this work, we sought to reverse engineer LBs and dissect the spatiotemporal events involved in their biogenesis at the genetic, molecular, biochemical, structural, and cellular levels. Toward this goal, we took advantage of a seeding-based model of -syn fibril formation in primary neurons and further developed this model to generate the first neuronal model that reproduces the key events leading to LB formation; including seeding, fibrillization, and the formation of LB-like inclusions that recapitulate many of the biochemical, structural, and organizational features of LBs found in post-mortem human PD brain tissues. Next, we applied an integrative approach combining confocal and correlative light-electron microscopy (CLEM) imaging methods with biochemical profiling of -syn species and temporal proteomic and transcriptomic analyses to dissect the molecular events associated with LB formation and maturation and to elucidate their contributions to neuronal dysfunctions and neurodegeneration in PD and synucleinopathies. The results from these studies demonstrate that LB formation involves a complex interplay between -syn fibrillization, post-translational modifications, and interactions between -syn aggregates and membranous organelles, including mitochondria and the autophagosome and endolysosome. Furthermore, we demonstrate that the process of LB formation and maturation, rather than simply fibril formation, is the major driver of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, as well as synaptic dysfunctions. Having a neuronal model that allows for unlinking of the key processes involved in LB formation is crucial for elucidating the molecular and cellular determinants of each process and their contributions to neuronal dysfunction and degeneration in PD and synucleinopathies. Such a model is essential to efforts to identify and investigate the mode of action and toxicity of drug candidates targeting -syn aggregation and LB formation.

neuroscience↗

bioRxiv2019

GCIB-SEM: A path to 10 nm isotropic imaging of cubic millimeter volumes

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) generates 3D datasets optimally suited for segmentation of cell ultrastructure and automated connectome tracing but is limited to small fields of view and is therefore incompatible with the new generation of ultrafast multibeam SEMs. In contrast, section-based techniques are multibeam-compatible but are limited in z-resolution making automatic segmentation of cellular ultrastructure difficult. Here we demonstrate a novel 3D electron microscopy technique, Gas Cluster Ion Beam SEM (GCIB-SEM), in which top-down, wide-area ion milling is performed on a series of thick sections, acquiring < 10 nm isotropic datasets of each which are then stitched together to span the full sectioned volume. Based on our results, incorporating GCIB-SEM into existing single beam and multibeam SEM workflows should be straightforward and should dramatically increase reliability while simultaneously improving z-resolution by a factor of 3 or more.

neuroscience↗

bioRxiv2018

The making of a Lewy body: the role of alpha-synuclein post-fibrillization modifications in regulating the formation and the maturation of pathological inclusions.

Although converging evidence point to alpha-synuclein (a-syn) aggregation and Lewy body (LB) formation as central events in Parkinson's disease (PD), the molecular mechanisms that regulate these processes and their role in disease pathogenesis remain poorly understood. Herein, we applied an integrative biochemical, structural and imaging approach to elucidate the sequence, molecular and cellular mechanisms that regulate LB formation in primary neurons. Our results establish that post-fibrillization C-terminal truncation mediated by calpains 1 and 2 and potentially other enzymes, plays critical roles in regulating a-syn seeding, fibrillization and orchestrates many of the events associated with LB formation and maturation. These findings combined with the abundance of a-syn truncated species in LBs and pathological a-syn aggregates have significant implications for ongoing efforts to develop therapeutic strategies based on targeting the C-terminus of a-syn or proteolytic processing of this region.

neuroscience↗

bioRxiv2017

Molecular insights into Vibrio cholerae’s intra-amoebal host-pathogen interactions

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the hosts osmoregulatory organelle. In this study, we deciphered the molecular mechanisms involved in the maintenance of V. choleraes intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrated that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, play a key role role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

microbiology↗