Biology subjects

Klenerman, D.

Publications and source records attributed to Klenerman, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A human neuronal model of sporadic Alzheimer's disease induced by FBXO2 downregulation shows A β aggregation, tau hyperphosphorylation and functional network impairment

Sporadic Alzheimers disease (sAD) arises from a complex interplay between genetic and environmental factors that remains poorly understood, making it challenging to develop accurate cell models. To address this problem, by hypothesing that the early disease sAD states can be characterised by transcriptomic fingerprints, we assessed the effect on A{beta} aggregation in human neuroblastoma cells a set of genes obtained by analysing snRNA-seq data from post-mortem AD patients. We then validated the most effective genes in human iPSC-derived cortical neurons, and selected FBXO2, a gene encoding a subunit of the ubiquitin protein ligase complex SCF, for further analysis. We found that early downregulation of FBXO2 in human iPSC-derived cortical neurons resulted in A{beta} aggregation, tau hyperphosphorylation, and structural and functional neuronal network impairment. Based on these results, we report a neuronal sAD model (FBXO2 KD sAD) that recapitulates a set of molecular hallmarks of sAD. We suggest that this strategy can be expanded towards the generation of panels of preclinical stem cell-derived models that recapitulate the molecular complexity of the broad spectrum of AD patients.

neuroscience↗

bioRxiv2024

Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells glycocalyx is much deeper than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation and reduced cytotoxicity, were observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes, increasing the killing of CRC cells. Similar enhanced interaction was observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

immunology↗

bioRxiv2024

SynPull: a novel method for studying neurodegeneration-related aggregates in synaptosomes using super-resolution microscopy

Synaptic dysfunction is one of the primary hallmarks of both Alzheimers and Parkinsons disease, leading to cognitive and behavioural decline. While alpha-synuclein, beta-amyloid, and tau are involved in the physiological functioning of synapses, their pathological aggregation has been linked to synaptic dysfunction. However, the methodology for studying the small (sub-diffraction limit) and soluble aggregates -often called oligomers, formed by these proteins is limited. Here we describe SynPull, a novel method combining single-molecule pulldown, super-resolution microscopy, and advanced computational analyses, in order to reliably study the quantity and morphology of the oligomeric alpha-synuclein, beta-amyloid, and AT8-positive tau aggregates in synaptosomes harvested from post-mortem human brain samples and mouse models. Using SynPull, we show that AT8-positive tau is the predominant aggregate type in AD, with significantly more aggregates compared to the control samples, yet the aggregate size does not differ between disease and control samples. Meanwhile, the relatively smaller amount of alpha-synuclein and beta-amyloid aggregates found in the synapses are larger than the extra-synaptic ones. Collectively, these results show the utility of SynPull to study pathological aggregates in dementia, which can help further understand the disease mechanisms causing synaptic dysfunction. O_FIG O_LINKSMALLFIG WIDTH=140 HEIGHT=200 SRC="FIGDIR/small/609517v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@d53d06org.highwire.dtl.DTLVardef@1995971org.highwire.dtl.DTLVardef@bf3edaorg.highwire.dtl.DTLVardef@1b75ca5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Human post-mortem orbitofrontal cortex samples from subjects with neuropathological diagnosis of Alzheimers and Parkinsons disease, as well as age-matched controls cut into [~]300 mg sections, and MI2, APPNL-G-F, P301S, and C57Bl/6J mouse brains were first homogenised in synaptosome buffer using a Dounce homogeniser and then filtered and centrifuged to separate nuclei and organelles from the synaptic fragments. Then, the isolated synaptosomes were incubated on the SiMPull surface with anti-neurexin antibody overnight, followed by fixation and permeabilisation. Imaging antibodies against beta-amyloid, alpha-synuclein, and AT8-positive tau were added to the samples and dSTORM imaging was performed to super-resolve the aggregates. C_FIG

neuroscience↗

bioRxiv2024

Selective suppression of oligodendrocyte-derived amyloid beta rescues neuronal dysfunction in Alzheimer's Disease

Reduction of amyloid beta (A{beta}) has been shown to be effective in treating Alzheimers Disease (AD), but the underlying assumption that neurons are the main source of pathogenic A{beta} is untested. Here we challenge this prevailing belief by demonstrating that oligodendrocytes are an important source of A{beta}, and play a key role in promoting abnormal neuronal hyperactivity in AD. We show that selectively suppressing oligodendrocyte A{beta} production improves AD brain pathology and restores neuronal function in vivo. Our findings suggest that targeting oligodendrocyte A{beta} production could be a promising therapeutic strategy for treating AD.

neuroscience↗

bioRxiv2023

The delayed kinetics of Myddosome formation explains why Aβ aggregates trigger TLR4 less efficiently than LPS

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 signals via the Myddosome when triggered by Lipopolysaccharide (LPS) or Amyloid-Beta (A{beta}) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated A{beta} 1-42 fibrils (80 seconds vs 372 seconds). The mean lifetimes of the Myddosomes was also shorter when triggered by LPS compared to sonicated A{beta} fibrils (170 and 220 s) respectively. In both cases a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively our data suggests that compared to LPS the multivalency of A{beta} fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated A{beta} fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

biophysics↗

bioRxiv2023

Characterization and super-resolution imaging of small tau aggregates in human samples.

Hyperphosphorylation and aggregation of the microtubule binding protein tau plays a key role in the development of Alzheimers disease. While the structure of the filamentous aggregates formed in humans has recently been determined to atomic resolution, there is far less information available about the smaller aggregate precursors, thought to be the most neurotoxic. To address this gap, we have developed a single molecule pull-down (SiMPull) able to detect tau aggregates in clinically relevant human samples. This method enables the detection and characterisation of individual tau aggregates, as opposed to averaged features obtained from traditional bulk techniques. We report the number, size and shape of individual aggregates measured via super-resolution microscopy, revealing disease-specific differences in tau aggregate morphology. By adapting the assay to simultaneously detect multiple phosphorylation sites in individual aggregates, we were also able to derive compositional profiles for pathological modifications present in individual aggregates. We demonstrate that tau aggregates in Alzheimers disease are significantly more likely to contain both the AT8 and T181 pathological phosphorylation markers, rather than only one. Together, tau SiMPull identified distinct subpopulations of large, modified tau aggregates that were invisible to traditional methodologies. These morphological and compositional differences distinguish samples taken from disease cohorts, offering to illuminate underlying disease mechanisms, and providing a foundation for novel diagnostic strategies.

biochemistry↗

bioRxiv2023

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 {+/-} 0.1%, 0.1 locs m-2) and sensitivity (>86.6 {+/-} 0.9%, 0.1 locs m-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs m-2) in dense cytosolic tubulin datasets.

biophysics↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2022

Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains

Neurodegenerative diseases, such as Alzheimers Disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low molecular weight aggregates play a key role in disease-associated toxicity. Within these aggregates, protofibrillar loop-like structures have been observed for a variety of amyloid systems and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils has largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterise amyloid ring structures derived from the brains of AD patients. We analyse the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. Furthermore, we show that these findings can be extended to several amyloid systems, giving a general framework relating the mechanical properties of assemblies and the conditions in which they can form loop structures. These results explain the diversity in the structures formed from protein aggregation and sheds light on the links between early forms of flexible ring-forming aggregates and their role in disease.

molecular biology↗

bioRxiv2022

Structural conversion of α-synuclein at the mitochondria induces neuronal toxicity

Aggregation of -Synuclein (-Syn) drives Parkinsons disease, although the initial stages of self-assembly and structural conversion have not been captured inside neurons. We track the intracellular conformational states of -Syn utilizing a single-molecule FRET biosensor, and show that -Syn converts from its monomeric state to form two distinct oligomeric states in neurons in a concentration dependent, and sequence specific manner. 3D FRET-CLEM reveals the structural organization, and location of aggregation hotspots inside the cell. Notably multiple intracellular seeding events occur preferentially on membrane surfaces, especially mitochondrial membranes. The mitochondrial lipid, cardiolipin triggers rapid oligomerization of A53T -Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial ROS generation, which accelerates the oligomerization of A53T -Syn, and ultimately causes permeabilization of mitochondrial membranes, and cell death. Patient iPSC derived neurons harboring A53T mutations exhibit accelerated oligomerization that is dependent on mitochondrial ROS, early mitochondrial permeabilization and neuronal death. Our study highlights a mechanism of de novo oligomerization at the mitochondria and its induction of neuronal toxicity.

neuroscience↗

bioRxiv2022

Uncovering the universality of self-replication in protein aggregation and its link to disease.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

biophysics↗

bioRxiv2022

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

biophysics↗

bioRxiv2021

AN ECONOMIC, SQUARE-SHAPED FLAT-FIELD ILLUMINATION MODULE FOR TIRF-BASED SUPER-RESOLUTION MICROSCOPY

Super-resolution (SR) microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard-to-implement, or not applicable to total internal reflection fluorescence (TIRF) imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fibre as the coupler. We characterise our method with synthetic, recombinant and cellular systems imaged under TIRF and highly inclined and laminated optical sheet (HILO) illuminations to demonstrate its ability to produce highly uniform images under all conditions.

biophysics↗

bioRxiv2021

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer's disease

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimers Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-{beta} (A{beta}) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with A{beta} in the early stages of aggregation and then fall away as fibrillation happens. ApoE-A{beta} co-aggregates account for [~]50% of the mass of soluble A{beta} aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of A{beta}. Selectively removing non-lipidated apoE4-A{beta} co-aggregates enhances clearance of toxic A{beta} by glial cells, and reduces inflammation and membrane damage, demonstrating a clear path to AD therapeutics.

neuroscience↗

bioRxiv2020

Construction of a liquid-liquid phase separation system from the gel-sol transition of elongated protein microgels in a crowding agent

Liquid proteinaceous materials have been frequently found in cells or tissues and are crucial for various biological processes. Unlike their solid-state counterparts, liquid-state protein compartments are challenging to engineer and control at the microscale. Conventionally, gelation (sol-gel transition) of biological molecules has been thought to be the intermediate step between liquid-liquid phase separation (LLPS) states and insoluble aggregates that are related to protein functions, malfunctions and even diseases. However, the opposite process, i.e., the gel-sol transition of materials, has not been broadly explored. Here we describe a thermoresponsive gel-sol transition of a protein in a crowded environment that results in a demixed LLPS state, contradicting the common consequence of a one-phase protein solution by the end of such transition at elevated temperature without crowding agents. We also demonstrate a simple method to monitor the gel-sol transition by showing that elongated gelatin microgels can evolve towards a spherical morphology in the crowding agents because of interfacial tension. The LLPS system was explored for the diffusion of small particles for drug-release application scenarios. Our results demonstrate a route for the rapid construction of LLPS models, where the gel-sol transition of the protein-rich phase is monitorable. The models are featured with tunable size and dimensional monodispersity of dispersed condensates. The present study can be employed in biophysics and bioengineering with practices such as 3D printing and temperature sensing.

biophysics↗

bioRxiv2020

Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, while the Alzheimer's R47H mutant does the opposite

Missense mutations (e.g. R47H) of the microglial receptor TREM2 increase risk of Alzheimers disease (AD), and the soluble ectodomain of wild-type TREM2 (sTREM2) appears to protect in vivo, but the underlying mechanisms are unclear. We show that A{beta} oligomers bind to TREM2, inducing shedding of sTREM2. Wild-type sTREM2 inhibits A{beta} oligomerization, fibrillization and neurotoxicity, and disaggregates preformed A{beta} oligomers and protofibrils. In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric A{beta}, but rather promotes A{beta} protofibril formation and neurotoxicity. Thus, in addition to mediating phagocytosis, wild-type TREM2 may protect against amyloid pathology by A{beta}-induced release of sTREM2 that blocks A{beta} aggregation and neurotoxicity; while R47H sTREM2 promotes A{beta} aggregation into neurotoxic forms, which may explain why the R47H variant gene increases AD risk several fold.

biochemistry↗

bioRxiv2020

Amplification, not spreading limits rate of tau aggregate accumulation in Alzheimer's disease

Both the replication of protein aggregates and their spreading throughout the brain are implicated in the progression of Alzheimers disease (AD). However, the rates of these processes are unknown and the identity of the rate-determining process in humans has therefore remained elusive. By bringing together chemical kinetics with measurements of tau seeds and aggregates across brain regions, we are able to quantify their replication rate in human brains. Remarkably, we obtain comparable rates in several different datasets, with 5 different methods of tau quantification, from seed amplification assays in vitro to tau PET studies in living patients. Our results suggest that the overall rate of accumulation of tau in neocortical regions is limited not by spreading between brain regions but by local replication, which doubles the number of seeds every ~5 years. Thus, we propose that limiting local replication constitutes the most promising strategy to control tau accumulation during AD.

biophysics↗

bioRxiv2020

Single-molecule light-sheet microscopy with local nanopipette delivery

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2{micro}m thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-{beta}, which causes proteotoxicity relevant to Alzheimers disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells. Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instruments capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimers disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

biophysics↗