O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology.

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.

Effective Inhibition of TDP-43 Aggregation by Native State Stabilization.

Preventing the misfolding or aggregation of transactive response DNA binding protein with 43 kDa (TDP-43) is the most actively pursued disease-modifying strategy to treat amyotrophic lateral sclerosis and other neurodegenerative diseases. In this work, we provide proof of concept that native state stabilization of TDP-43 is a viable and effective strategy for treating TDP-43 proteinopathies. Firstly, we leveraged the Cryo-EM structures of TDP-43 fibrils to design C-terminal substitutions that disrupt TDP-43 aggregation. Secondly, we showed that these substitutions (S333D/S342D) stabilize monomeric TDP-43 without altering its physiological properties. Thirdly, we demonstrated that binding native oligonucleotide ligands stabilized monomeric TDP-43 and prevented its fibrillization and phase separation in the absence of direct binding to the aggregation-prone C-terminal domain. Fourthly, we showed that the monomeric TDP-43 variant could be induced to aggregate in a controlled manner, which enabled the design and implementation of a high-throughput screening assay to identify native state stabilizers of TDP-43. Altogether, our findings demonstrate that different structural domains in TDP-43 could be exploited and targeted to develop drugs that stabilize the native state of TDP-43 and provide a platform to discover novel drugs to treat TDP-43 proteinopathies.

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 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 α-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 α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases.

The abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. Various aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance among these disorders. Relying on antibodies that have not been assessed for their ability to detect the diverse forms of aSyn may lead to inaccurate estimations of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterized an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn, and that recognizes all monomeric, oligomeric, and fibrillar aSyn conformations. Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneous forms of aSyn pathology, rich in Serine 129 phosphorylation, Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered both to neurons and glia. In addition, we show that aSyn can become hyperphosphorylated during processes of aggregation and inclusion maturation in neuronal and animal models of aSyn seeding and spreading. The validation pipeline we describe for these antibodies paves the way for systematic investigations into aSyn pathological diversity in the human brain, peripheral tissues, as well as in cellular and animal models of synucleinopathies.

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 Huntington's disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to 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. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information.

Overexpression of human alpha-Synuclein leads to dysregulated microbiome/metabolites with ageing in a rat model of Parkinson disease.

BACKGROUND: Braak's hypothesis states that sporadic Parkinson's disease (PD) follows a specific progression of pathology from the peripheral to the central nervous system, and this progression can be monitored by detecting the accumulation of alpha-Synuclein (α-Syn) protein. Consequently, there is growing interest in understanding how the gut (commensal) microbiome can regulate α-Syn accumulation, as this could potentially lead to PD. METHODS: We used 16S rRNA and shotgun sequencing to characterise microbial diversity. 1H-NMR was employed to understand the metabolite production and intestinal inflammation estimated using ELISA and RNA-sequencing from feces and the intestinal epithelial layer respectively. The Na+ channel current and gut permeability were measured using an Ussing chamber. Immunohistochemistry and immunofluorescence imaging were applied to detect the α-Syn protein. LC-MS/MS was used for characterization of proteins from metabolite treated neuronal cells. Finally, Metascape and Ingenuity Pathway Analysis (IPA) bioinformatics tools were used for identification of dysregulated pathways. RESULTS: We studied a transgenic (TG) rat model overexpressing the human SNCA gene and found that a progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young TG rats. Interestingly, this ratio then increased with ageing. The dynamics of Lactobacillus and Alistipes were monitored and reduced Lactobacillus and increased Alistipes abundance was discerned in ageing TG rats. Additionally, the SNCA gene overexpression resulted in gut α-Syn protein expression and increased with advanced age. Further, older TG animals had increased intestinal inflammation, decreased Na+ current and a robust alteration in metabolite production characterized by the increase of succinate levels in feces and serum. Manipulation of the gut bacteria by short-term antibiotic cocktail treatment revealed a complete loss of short-chain fatty acids and a reduction in succinate levels. Although antibiotic cocktail treatment did not change α-Syn expression in the enteric nervous system of the colon, however, reduced α-Syn expression was detected in the olfactory bulbs (forebrain) of the TG rats. CONCLUSION: Our data emphasize that the gut microbiome dysbiosis synchronous with ageing leads to a specific alteration of gut metabolites and can be modulated by antibiotics which may affect PD pathology.

Fully co-factor-free ClearTau platform produces seeding-competent Tau fibrils for reconstructing pathological Tau aggregates.

Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.

Artificial intelligence-coupled plasmonic infrared sensor for detection of structural protein biomarkers in neurodegenerative diseases.

Diagnosis of neurodegenerative disorders (NDDs) including Parkinson's disease and Alzheimer's disease is challenging owing to the lack of tools to detect preclinical biomarkers. The misfolding of proteins into oligomeric and fibrillar aggregates plays an important role in the development and progression of NDDs, thus underscoring the need for structural biomarker-based diagnostics. We developed an immunoassay-coupled nanoplasmonic infrared metasurface sensor that detects proteins linked to NDDs, such as alpha-synuclein, with specificity and differentiates the distinct structural species using their unique absorption signatures. We augmented the sensor with an artificial neural network enabling unprecedented quantitative prediction of oligomeric and fibrillar protein aggregates in their mixture. The microfluidic integrated sensor can retrieve time-resolved absorbance fingerprints in the presence of a complex biomatrix and is capable of multiplexing for the simultaneous monitoring of multiple pathology-associated biomarkers. Thus, our sensor is a promising candidate for the clinical diagnosis of NDDs, disease monitoring, and evaluation of novel therapies.

Seeding the aggregation of TDP-43 requires post-fibrillization proteolytic cleavage.

Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a ß-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.

Neuronal growth on high-aspect-ratio diamond nanopillar arrays for biosensing applications.

Monitoring neuronal activity with simultaneously high spatial and temporal resolution in living cell cultures is crucial to advance understanding of the development and functioning of our brain, and to gain further insights in the origin of brain disorders. While it has been demonstrated that the quantum sensing capabilities of nitrogen-vacancy (NV) centers in diamond allow real time detection of action potentials from large neurons in marine invertebrates, quantum monitoring of mammalian neurons (presenting much smaller dimensions and thus producing much lower signal and requiring higher spatial resolution) has hitherto remained elusive. In this context, diamond nanostructuring can offer the opportunity to boost the diamond platform sensitivity to the required level. However, a comprehensive analysis of the impact of a nanostructured diamond surface on the neuronal viability and growth was lacking. Here, we pattern a single crystal diamond surface with large-scale nanopillar arrays and we successfully demonstrate growth of a network of living and functional primary mouse hippocampal neurons on it. Our study on geometrical parameters reveals preferential growth along the nanopillar grid axes with excellent physical contact between cell membrane and nanopillar apex. Our results suggest that neuron growth can be tailored on diamond nanopillars to realize a nanophotonic quantum sensing platform for wide-field and label-free neuronal activity recording with sub-cellular resolution.

O-GlcNAc modification forces the formation of an α-Synuclein amyloid-strain with notably diminished seeding activity and pathology.

The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such α-Synuclein and Tau, which are implicated in the pathogenesis of Alzheimer's and Parkinson's disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies. In this work, we report that O-GlcNAc modification of α-Synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by Cryo-EM, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-Synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that post-translational modifications, such as O-GlcNAc modification, of α-Synuclein are key determinants of α-Synuclein amyloid strains and pathogenicity. These findings have significant implications for how we investigate and target amyloids in the brain and could possibly explain the lack of correlation between amyloid burden and neurodegeneration or cognitive decline in some subtypes of NDDs.

Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein.

Cell-to-cell transmission and subsequent amplification of pathological proteins promote neurodegenerative disease progression. Most research on this has focused on pathological protein seeds, but how their normal counterparts, which are converted to pathological forms during transmission, regulate transmission is less understood. Here we show in cultured cells that phosphorylation of soluble, nonpathological α-synuclein (α-Syn) at previously identified sites dramatically affects the amplification of pathological α-Syn, which underlies Parkinson's disease and other α-synucleinopathies, in a conformation- and phosphorylation site-specific manner. We performed LC-MS/MS analyses on soluble α-Syn purified from Parkinson's disease and other α-synucleinopathies, identifying many new α-Syn post-translational modifications (PTMs). In addition to phosphorylation, acetylation of soluble α-Syn also modified pathological α-Syn transmission in a site- and conformation-specific manner. Moreover, phosphorylation of soluble α-Syn could modulate the seeding properties of pathological α-Syn. Our study represents the first systematic analysis how of soluble α-Syn PTMs affect the spreading and amplification of pathological α-Syn, which may affect disease progression.

Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders.

Alpha-synuclein (aSyn) is a pre-synaptic monomeric protein that can form aggregates in neurons in Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB), and in oligodendrocytes in multiple system atrophy (MSA). Although aSyn in astrocytes has previously been described in PD, PDD and DLB, the biochemical properties and topographical distribution of astrocytic aSyn have not been studied in detail. Here, we present a systematic investigation of aSyn astrocytic pathology using an expanded antibody toolset covering the entire sequence and key post-translational modifications (PTMs) of aSyn in Lewy body disorders (LBDs) and in MSA. Astrocytic aSyn was detected in the limbic cortical regions of LBDs but were absent in main pathological regions of MSA. The astrocytic aSyn was revealed only with antibodies against the mid N-terminal and non-amyloid component (NAC) regions covering aSyn residues 34-99. The astroglial accumulations were negative to canonical aSyn aggregation markers, including p62, ubiquitin and aSyn pS129, but positive for phosphorylated and nitrated forms of aSyn at Tyrosine 39 (Y39), and not resistant to proteinase K. Our findings suggest that astrocytic aSyn accumulations represent a major part of aSyn pathology in LBDs and possess a distinct sequence and PTM signature that is characterized by both N- and C-terminal truncations and modifications at Y39. This is the first description that aSyn accumulations are made solely from N- and C-terminally cleaved aSyn species and the first report demonstrating that astrocytic aSyn is a mixture of Y39 phosphorylated and nitrated species. These observations underscore the importance of systematic characterization of aSyn accumulations in different cell types to capture the aSyn pathological diversity in the brain. Our findings combined with further studies on the role of astrocytic pathology in the progression of LBDs can pave the way towards identifying novel disease mechanisms and therapeutic targets.

N-terminal mutant huntingtin deposition correlates with CAG repeat length and symptom onset, but not neuronal loss in Huntington's disease.

Huntington's disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake an immunohistochemical screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabeled human brain tissue. Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with a customized automated image analysis pipeline on the TMAs. A 2.5-12 fold increase in the number of Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. MAB5374, MW1, and EPR5526 Htt aggregate numbers were positively correlated with CAG repeat length, and negatively correlated with the age of symptom onset in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and greater Htt cortical aggregate deposition is associated with an earlier age of symptom onset in HD. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD do not specifically immunolabel Htt aggregates in HD human brain tissue exclusively, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity.

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson's disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.

Pathological substrate of memory impairment in multiple system atrophy.

AIMS: Synaptic dysfunction in Parkinson's disease is caused by propagation of pathogenic α-synuclein between neurons. Previously, in multiple system atrophy (MSA), pathologically characterised by ectopic deposition of abnormal α-synuclein predominantly in oligodendrocytes, we demonstrated that the occurrence of memory impairment was associated with the number of α-synuclein-positive neuronal cytoplasmic inclusions (NCIs) in the hippocampus. In the present study, we aimed to investigate how abnormal α-synuclein in the hippocampus can lead to memory impairment. METHODS: We performed pathological and biochemical analyses using a mouse model of adult-onset MSA and human cases (MSA, N = 25; Parkinson's disease, N = 3; Alzheimer's disease, N = 2; normal controls, N = 11). In addition, the MSA model mice were examined behaviourally and physiologically. RESULTS: In the MSA model, inducible human α-synuclein was first expressed in oligodendrocytes and subsequently accumulated in the cytoplasm of excitatory hippocampal neurons (NCI-like structures) and their presynaptic nerve terminals with the development of memory impairment. α-Synuclein oligomers increased simultaneously in the hippocampus of the MSA model. Hippocampal dendritic spines also decreased in number, followed by suppression of long-term potentiation. Consistent with these findings obtained in the MSA model, post-mortem analysis of human MSA brain tissues showed that cases of MSA with memory impairment developed more NCIs in excitatory hippocampal neurons along with α-synuclein oligomers than those without. CONCLUSIONS: Our results provide new insights into the role of α-synuclein oligomers as a possible pathological cause of memory impairment in MSA.

Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson's disease and other synucleinopathies.

Parkinson's disease (PD), the second most common progressive neurodegenerative disease, develops and progresses for 10-15 years before the clinical diagnostic symptoms of the disease are manifested. Furthermore, several aspects of PD pathology overlap with other neurodegenerative diseases (NDDs) linked to alpha-synuclein (aSyn) aggregation, also called synucleinopathies. Therefore, there is an urgent need to discover and validate early diagnostic and prognostic markers that reflect disease pathophysiology, progression, severity, and potential differences in disease mechanisms between PD and other NDDs. The close association between aSyn and the development of pathology in synucleinopathies, along with the identification of aSyn species in biological fluids, has led to increasing interest in aSyn species as potential biomarkers for early diagnosis of PD and differentiate it from other synucleinopathies. In this review, we (1) provide an overview of the progress toward mapping the distribution of aSyn species in the brain, peripheral tissues, and biological fluids; (2) present comparative and critical analysis of previous studies that measured total aSyn as well as other species such as modified and aggregated forms of aSyn in different biological fluids; and (3) highlight conceptual and technical gaps and challenges that could hinder the development and validation of reliable aSyn biomarkers; and (4) outline a series of recommendations to address these challenges. Finally, we propose a combined biomarker approach based on integrating biochemical, aggregation and structure features of aSyn, in addition to other biomarkers of neurodegeneration. We believe that capturing the diversity of aSyn species is essential to develop robust assays and diagnostics for early detection, patient stratification, monitoring of disease progression, and differentiation between synucleinopathies. This could transform clinical trial design and implementation, accelerate the development of new therapies, and improve clinical decisions and treatment strategies.