Biology subjects

Lashuel, H.

Publications and source records attributed to Lashuel, H..

4 recordsLinked to original sources

bioRxiv2020

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimers disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in {beta}-sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.

biochemistry↗

bioRxiv2019

A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: towards improving experimental reproducibility in α-synuclein research

Increasing evidence suggests that the process of alpha-synuclein (aSyn) aggregation from monomers into amyloid fibrils via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinsons disease (PD), multiple system atrophy and dementia with Lewy bodies. However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying the putative toxic species along the pathway of aSyn fibrillization, and to develop small molecule drugs or antibodies that target toxic aSyn oligomeric intermediates. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different aSyn species, many of the fundamental questions regarding the role of aSyn aggregation in PD remain unanswered, and no therapeutic compounds targeting aSyn oligomers have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of aSyn aggregates. In the majority of experiment, the aSyn samples used contain mixtures of aSyn species that exist in an equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different aSyn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinsons research, we describe here a simple centrifugation-based filtration protocol for the isolation, quantification and assessment of the distribution of of aSyn monomers, oligomers and fibrils, in heterogeneous aSyn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of aSyn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid-forming proteins.\n\nTable of Content Figure\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC=\"FIGDIR/small/772160v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (33K):\norg.highwire.dtl.DTLVardef@13fb942org.highwire.dtl.DTLVardef@9bc3adorg.highwire.dtl.DTLVardef@4b9c66org.highwire.dtl.DTLVardef@9054f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM

The misfolding and self-assembly of proteins into {beta}-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo-EM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-{beta}, tau, -synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin {lambda} light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo-EM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo-EM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimers disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo-EM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a powerful tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. These advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

neuroscience↗

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↗