Biology subjects

Lashuel, H.

Publications and source records attributed to Lashuel, H..

At least 19 recordsLinked to original sources

bioRxiv2026

WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease

The aggregation of -synuclein (-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinsons disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding -SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of -SYN assembly in neurons. We found that the initiation and accumulation of -SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the -SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo -SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances -SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated -SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of -SYN oligomerization in living neurons and identify the WDR44--SYN interaction as a promising therapeutic target for reducing -SYN pathology and enabling early intervention in PD.

neuroscience↗

bioRxiv2025

A Site-Specific Organometallic Approach for Installing Tyrosine Phosphorylation Mimics to Decipher the Role of Phosphorylation in Alpha-Synuclein (aSyn) Aggregation and Seeding

Several studies have identified phosphorylation of alpha-synuclein (aSyn) at multiple tyrosine residues (Y39, Y125, Y133, and Y136) within Lewy bodies, which are the pathological hallmarks of Parkinsons disease (PD). However, understanding the specific role of phosphorylation at each site, or how multiple phosphorylation sites interact, has been challenging. Herein, we present an efficient method that leverages an organometallic Pd-complex to site-specifically attach phosphomimetic groups via Cys-arylation, closely mimicking natural phosphorylation. Using this approach, we successfully incorporated native-like tyrosine phosphorylation mimics into cysteine-containing proteins like synthetic transcription factor Max and recombinant aSyn, in good yields. To demonstrate the methods versatility, we created a focused library of mono-, di-, and tri-phosphorylated aSyn analogues. This development enabled, for the first time, the investigation of the effect of site-specific phosphorylation at multiple C-terminal tyrosine residues on aSyn fibrillization and aggregation. Our results show that mono-phosphorylation at any of the C-terminal sites has little effect on aggregation, while di- and tri-phosphorylation slows down the process, extending the lag phase compared to wild-type aSyn. Additionally, phosphorylation at Y39 reduces the seeding activity of aSyn fibrils by almost threefold in both mammalian and neuronal models of synuclein pathology. Overall, our strategy provides a rapid, selective, and scalable way to introduce aromatic PTMs, producing homogeneous protein libraries essential for mechanistic studies, biomarker development, and exploring the therapeutic potential of targeting aSyn-phosphorylation for PD and related disorders.

biochemistry↗

bioRxiv2025

Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington's disease human cerebral cortex

Huntingtons disease (HD) is a hereditary neurodegenerative condition caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein. The accumulation of Htt inclusion bodies is a pathological hallmark of HD and a common target for therapeutic strategies. However, the limited efficacy of treatments targeting the Htt protein highlights the need for a better understanding of the role of Htt inclusion bodies in HD pathogenesis. This study examined the heterogeneity of Htt inclusion body composition by co-labelling with three Htt epitope-specific antibodies to characterize Htt inclusion body immunophenotype. We then characterized the size and sub-cellular location of Htt inclusions with distinct immunophenotypes. Using multiplex immunohistochemistry, we also examined the ubiquitination profile of each immunophenotype. Our findings demonstrate that Htt inclusions have a range of immunophenotypes, with some labelled by only one of the three antibodies and others exhibiting co-labelling by several antibodies, thus demonstrating the heterogeneity in inclusion composition and structure. We outline evidence that inclusion bodies exclusively labelled with the EM48 antibody are small, non-nuclear, and more abundant in HD cases with increased CAG repeat length, higher Vonsattel grade, and earlier age of onset. We also find that Htt inclusion bodies labelled by multiple antibodies are more likely to be ubiquitinated, predominantly by K63-rather than K48-linked ubiquitin, suggesting preferential degradation by autophagy. Lastly, we show that ubiquitinated Htt inclusion bodies are more highly immunoreactive for ubiquilin 2 than p62. Our findings highlight the need for multiple antibodies to capture the full spectrum of Htt pathology in HD and imply that future studies should consider the diversity of inclusion body composition and structure when correlating pathology formation to neurodegeneration, clinical symptoms, or disease severity.

neuroscience↗

bioRxiv2024

Mouse α-synuclein fibrils are structurally and functionally distinct from human fibrils associated with Lewy body diseases

Short AbstractThe intricate process of -synuclein aggregation and fibrillization hold pivotal roles in Parkinsons disease (PD) and multiple system atrophy (MSA). While mouse -synuclein can fibrillize in vitro, whether these fibrils commonly used in research to induce this process or form can reproduce structures in the human brain remains unknown. Here we report the first atomic structure of mouse -synuclein fibrils, which was solved in parallel by two independent teams. The structure shows striking similarity to MSA-amplified and PD-associated E46K fibrils. However, mouse -synuclein fibrils display altered packing arrangements, reduced hydrophobicity, heightened fragmentation sensitivity, and evoke only weak immunological responses. Furthermore, mouse -synuclein fibrils exhibit exacerbated pathological spread in neurons and humanized -synuclein mice. These findings provide new insights into the structural underpinnings of -synuclein pathogenicity and emphasize a need to reassess the role of mouse -synuclein fibrils in the development of related diagnostic probes and therapeutic interventions.

neuroscience↗

bioRxiv2023

Differential Effects of Posttranslational Modifications on the Membrane Interaction of Huntingtin Protein

Huntingtons disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple posttranslational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the impact of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17 membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insight into the complex relationship between Nt17 structure, PTMs and membrane binding. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/569526v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@153610eorg.highwire.dtl.DTLVardef@10908dforg.highwire.dtl.DTLVardef@256c14org.highwire.dtl.DTLVardef@dac763_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Deep learning-assisted single-molecule detection of protein post-translational modifications with a biological nanopore

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both the spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish -synuclein-derived peptides bearing single or multiple PTMs, namely phosphorylation, nitration and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the -synuclein peptide and its PTM variants could be confidently identified using a deep learning model for signal processing. We further demonstrate that this framework can quantify -synuclein peptides at picomolar concentration and detect the C-terminal peptides generated by digestion of full-length -synuclein. Collectively, our work highlights the unique advantage of using nanopore as a tool for simultaneous detection of multiple PTMs and paves the way for their use in biomarker discovery and diagnostics.

biophysics↗

bioRxiv2023

Label-free Identification of Protein Aggregates Using Deep Learning

Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntingtons disease, which is caused by a genetic mutation that leads to a polyglutamine repeat length > 35 in exon 1 of the Huntingtin protein (Httex1). Current research on protein aggregation often involves the use of fluorescent labels to visualize and monitor the dynamics of protein expression, which can alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. We developed pixel-classification and pixel-regression models, which are robust across imaging conditions, and validated them on aggregates formed by different constructs of Httex1. Our results reveal that Httex1 proteins with shorter polyglutamine repeat lengths form aggregates with a higher average dry mass and area, highlighting the differences in their ultrastructure and aggregation mechanisms. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process. Our highly-robust models offer high speed, specificity, and simplicity to analyze label-free protein aggregation dynamics and obtain high-fidelity information.

bioengineering↗

bioRxiv2022

On demand nanoliter sampling probe for collection of brain fluid

Continuous fluidic sampling systems allow collection of brain biomarkers in vivo. Here, we propose a new sampling paradigm, Droplet on Demand (DoD), implemented in a microfabricated neural probe. It allows sampling droplets loaded with molecules from the brain extracellular fluid punctually, without the long transient equilibration periods typical of continuous methods. It uses an accurate fluidic sequence and correct operation is verified by the embedded electrodes. As a proof of concept, we demonstrated the application of this novel approach in vitro and in vivo, to collect glucose in the brain of mice, with a temporal resolution of 1-2 minutes and without transient regime. Absolute quantification of the glucose level in the samples was performed by direct infusion nanoelectrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS). By adjusting the diffusion time and the perfusion volume of DoD, the fraction of molecules recovered in the samples can be tuned to mirror the tissue concentration at accurate points in time. This makes quantification of biomarkers in the brain possible within acute experiments of only 20 to 120 minutes. DoD provides a complementary tool to continuous microdialysis and push-pull sampling probes. The advances allowed by DoD will benefit quantitative molecular studies in the brain, namely for molecules involved in volume transmission or for protein aggregates that form in neurodegenerative diseases over long periods. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/487549v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1edeb56org.highwire.dtl.DTLVardef@f573aforg.highwire.dtl.DTLVardef@7dfd38org.highwire.dtl.DTLVardef@17b7b46_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Comparative analysis of total alpha-synuclein (αSYN) immunoassays reveals that they do not capture the diversity of modified αSYN proteoforms

BackgroundThe development of therapeutics for Parkinsons disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the -synuclein (SYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. ObjectiveTo determine whether the three commercial kits that have been extensively used for total SYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant SYN proteoforms. MethodsWe investigated and compared the ability of the different assays to detect the diversity of SYN proteoform using a library of SYN proteins that compromise the majority of disease-relevant SYN variants and post-translational modification. ResultsOur findings showed that none of the three tested immunoassays accurately capture the totality of relevant SYN species and are unable to recognize most disease-associated C-terminally truncated variants of SYN. Moreover, several N-terminal truncations and phosphorylation/nitration differentially modify the level of SYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the SYN proteoforms analyzed by the three immunoassays. ConclusionsOur results showed that these immunoassays do not capture the totality of the relevant SYN species and therefore may not be appropriate tools to provide an accurate measure of total SYN levels in samples containing modified forms of the protein. This highlights the need for next-generation SYN immunoassays that capture the diversity of SYN proteoforms.

neuroscience↗

bioRxiv2021

Non-monotonic fibril surface occlusion by GFP tags from coarse-grained molecular simulations

The pathological growth of amyloid fibrils in neurons underlies the progression of neurodegenerative diseases including Alzheimers and Parkinsons disease. Fibrils form when soluble monomers oligomerise in the cytoplasm. Their subsequent growth occurs via nucleated polymerization mechanisms involving the free ends of the fibrils augmented by secondary nucleation of new oligomers at their surface. Amyloid fibrils possess a complex interactome with diffusing cytoplasmic proteins that regulates many aspects of their growth, seeding capacity, biochemical activity and transition to pathological inclusions in diseased brains. Changes to their surface are also expected to modify their interactome, pathogenicity and spreading in the brain. Many assays visualise fibril formation, growth and inclusion formation by decorating monomeric proteins with fluorescent tags such as GFP. Recent studies from our group suggest that tags with sizes comparable to the fibril radius may modify the fibril surface accessibility and thus their PTM pattern, interactome and ability to form inclusions. Using coarse-grained molecular simulations of a single alpha synuclein fibril tagged with GFP we find that thermal fluctuations of the tags create a non-monotonic, size-dependent sieve around the fibril that perturbs its interactome with diffusing species. Our results indicate that experiments using tagged and untagged monomers to study the growth and interactome of fibrils should be compared with caution, and the confounding effects of the tags are more complex than a reduction in surface accessibility. The prevalence of fluorescent tags in amyloid fibril growth experiments suggests this has implications beyond the specific alpha synuclein fibrils we model here.

biophysics↗

bioRxiv2021

The structural basis of huntingtin (Htt) fibril polymorphism, revealed by cryo-EM of exon 1 Htt fibrils

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein has hampered efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntingtons disease. To address this knowledge gap, we investigated the structural properties of in vitro generated fibrils from exon1 of the huntingtin protein by electron cryo-microscopy and single-particle analysis. We show that wildtype and mutant exon1 of the huntingtin protein form non-helical fibrils with a polygultamine amyloid core composed of {beta}-hairpins with unique characteristics that have not been previously observed with other amyloid filaments. The stacks of {beta}-hairpins form long planar {beta}- sheets (protofilaments) with variable stacking angle and occasional out-of-register state of individual {beta}-hairpins. These features and the propensity of protofilament to undergo lateral association results in a high degree of fibril polymorphism, including fibrils composed of varying numbers of protofilaments. Our results also represent the first direct observation of how the flanking domains are organized around the polyglutamine core of the fibril and provide insight into how they might affect huntingtin fibril structure, polymorphism, and stacking of {beta}-hairpins within its core structure. Removal of the first 17 amino acids at the N-terminus resulted in surprising intra-fibril structural heterogeneity and reduced fibrils propensity to lateral associations. Overall, this work provides valuable insights that could guide future mechanistic studies to elucidate the sequence and structural determinants of huntingtin aggregation, as well as cryo-EM and structural studies of fibrils derived from huntingtin proteins and other disease-associated polyglutamine-containing proteins.

neuroscience↗

bioRxiv2021

A new chemoenzymatic semisynthetic approach provides novel insight into the role of phosphorylation beyond exon1 of Huntingtin and reveals N-terminal fragment length-dependent distinct mechanisms of aggregation

Huntingtons disease is a neurodegenerative disorder caused by the expansion of a polyglutamine (poly Q) repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Htt exon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1. We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded novel insights into how PTMs and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slowed its aggregation, whereases phosphorylation at T107 and S116 accelerated the aggregation of Htt171, underscoring the importance of crosstalk between different PTMs. We demonstrate that mutant Htt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mutant Httex1. These observations suggest that different N-terminal fragments could have distinct mechanisms of aggregation and that a single polyQ-targeting anti-aggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the importance of further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity, pathology formation, and disease progression. Table of content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/436743v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@113aebborg.highwire.dtl.DTLVardef@1349236org.highwire.dtl.DTLVardef@6b273borg.highwire.dtl.DTLVardef@dcad5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Overexpression of human alpha-synuclein leads to dysregulated microbiome and metabolites with ageing in a rat model of Parkinson disease

Since Braaks hypothesis stating that sporadic Parkinsons disease follows a specific progression of the pathology from the peripheral to the central nervous system and can be monitored by detecting accumulation of the alpha-Synuclein protein. There is growing interest in understanding how the gut (commensal) microbiome can regulate alpha-Synuclein accumulation which can lead to PD. We studied a transgenic rat model overexpressing the human alpha-Synuclein and found that the protein overexpression resulted in gut alpha-Synuclein expression and aggregation in the gut neurons with advancing age. A progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young transgenic rat model and interestingly this ratio was then increased with aging. This observation was accompanied in older animals by intestinal inflammation, increase gut permeability and a robust alteration in metabolites production characterized by the increase of succinate level in the feces and serum. Manipulation of the gut bacteria by short-term antibiotics treatment revealed a complete loss of short-chain fatty acids (SCFAs) and reduction in succinate levels. Although antibiotics treatment did not change alpha-synuclein expression in the enteric nervous system of the colon, it can reduce alpha-synuclein expression in the olfactory bulb of the transgenic rats. In summary, synchronous with ageing, our data emphasize that the gut microbiome dysbiosis leads to a specific alteration of gut metabolites which are reflected in the serum and can be modulated by the environment.

neuroscience↗

bioRxiv2020

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the unfolded protein response (UPR) in models of neurodegeneration

Alteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinsons and Huntingtons disease. These results support the concept where directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

neuroscience↗

bioRxiv2020

Disentangling the sequence, cellular and ultrastructural determinants of Huntingtin nuclear and cytoplasmic inclusion formation

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntingtons disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide novel insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve 1) polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; 2) recruitment and accumulation of remodeled or dysfunctional membranous organelles, and 3) impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/226977v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@295056org.highwire.dtl.DTLVardef@594664org.highwire.dtl.DTLVardef@11c5384org.highwire.dtl.DTLVardef@af0e0a_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntingtin exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP, right).

neuroscience↗

bioRxiv2020

Functional and molecular early enteric biomarkers for Parkinson's disease in mice and men

Parkinsons disease (PD) usually has a late clinical onset. The lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use an -synuclein-overexpressing transgenic (Th-1-SNCA-A30P) mouse model of PD to identify appropriate candidate markers in the gut for early stages of PD before hallmark symptoms begin to manifest. A30P mice did not show alterations in gait parameters at 2 months of age, and these mice were therefore defined as pre-symptomatic A30P mice (psA30P). We discovered early functional motility changes in the gut and early molecular dysregulations in the myenteric plexus of psA30P mice by comparative protein and miRNA profiling and cell culture experiments. We found that the proteins neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are potential biomarkers of early PD that may facilitate timely treatment and/or prevention of PD in men.

neuroscience↗

bioRxiv2020

Monitoring alpha-synuclein oligomerization and inclusion formation using bimolecular fluorescence complementation assays: What you see is not always what you get.

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (-syn) oligomerization in intact cells. Since then, several -syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased -syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with -syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers of the fluorescent complex resemble oligomers formed on the pathway to -syn fibrillization. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of -syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of -syn upon co-expression of both BiFC fragments. Our results show that 1) the C-terminal-Venus fused to -syn (-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to -syn (Vn--syn) ; 2) Vn--syn exhibits a high propensity to form oligomers and higher-order aggregates; and 3) the expression of either or both fragments does not result in the formation of -syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn--syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of -syn-Vc in Vn--syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate -syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission that are based on the -syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/074161v3_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@fe4896org.highwire.dtl.DTLVardef@4f912forg.highwire.dtl.DTLVardef@1c4e157org.highwire.dtl.DTLVardef@a648e6_HPS_FORMAT_FIGEXP M_FIG C_FIG Bimolecular fluorescence complementation (BiFC) was introduced a decade ago to monitor alpha-synuclein oligomerization in intact cells, based on the assumption that an increase in the fluorescent signal correlates with -synuclein oligomerization and aggregation. Herein, we used several biochemical and cellular assays to characterize commonly used -synuclein Venus BiFC assays. Our results show that one of the BiFC fragments (Vn--synuclein) exhibits higher expression levels and aggregation propensity than its counterpart (-synuclein-Vc), thus complicating the interpretation of the molecular interactions that give rise to the fluorescence signal and raise concerns about their application to investigate -syn oligomerization in cells or in vivo.

biochemistry↗

bioRxiv2020

Pronounced α-synuclein pathology in a seeding-based mouse model is not sufficient to induce mitochondrial respiration deficits in the striatum and amygdala

Increasing evidence suggests that crosstalk between -synuclein pathology formation and mitochondrial dysfunctions plays a central role in the pathogenesis of Parkinsons disease and related synucleinopathies. While mitochondrial dysfunction is a well-studied phenomenon in the substantia nigra, which is selectively vulnerable in Parkinsons disease and some models thereof, less information is available in other brain regions that are also affected by synuclein pathology. Therefore, we sought to test the hypothesis that early -synuclein pathology causes mitochondrial dysfunction, and that this effect might be exacerbated in conditions of increased vulnerability of affected brain regions, such as the amygdala. We combined a model of intracerebral -synuclein pathology seeding with chronic glucocorticoid treatment modelling non-motor symptoms of Parkinsons disease and affecting amygdala physiology. We measured mitochondrial respiration, ROS generation and protein abundance as well as -synuclein pathology in male mice. Chronic corticosterone administration induced mitochondrial hyperactivity in the amygdala. Although injection of -synuclein preformed fibrils into the striatum resulted in pronounced -synuclein pathology in both striatum and amygdala, mitochondrial respiration in these brain regions was altered in neither chronic corticosterone nor control conditions. Our results suggest that early stage -synuclein pathology does not influence mitochondrial respiration in the striatum and amygdala, even in corticosterone-induced respirational hyperactivity. We discuss our findings in light of relevant literature, warn of a potential publication bias and encourage scientist to report their negative results in the frame of this model. Significance statementWe provide evidence that early stage synucleinopathy by itself or in combination with exogenous corticosterone induced amygdala hyperactivity did not compromise mitochondrial respiration in the striatum and amygdala in one of the most commonly used models of synucleinopathies. These results may explain, why this model in the hands of many research groups does not elicit pronounced Parkinsons disease like symptoms in the absence of mitochondrial dysfunction in brain regions strongly affected by synuclein pathology and involved in non-motor (amygdala) and motor (striatum) symptoms. Our findings call for rigorous investigation of the short- and long-term effects of -synuclein pathology on mitochondrial function/dysfunction in this model, in particular in brain regions strongly affected by neurodegeneration such as the substantia nigra pars compacta.

neuroscience↗