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.

Recombinant Full-Length TDP-43 Oligomers Retain Their Ability to Bind RNAs, Are Not Toxic, and Do Not Seed TDP-43 Aggregation in Vitro.

TAR DNA-binding protein with 43 kD (TDP-43) is a partially disordered protein that misfolds and accumulates in the brains of patients affected by several neurodegenerative diseases. TDP-43 oligomers have been reported to form due to aberrant misfolding or self-assembly of TDP-43 monomers. However, very little is known about the molecular and structural basis of TDP-43 oligomerization and the toxic properties of TDP-43 oligomers due to several reasons, including the lack of conditions available for isolating native TDP-43 oligomers or producing pure TDP-43 oligomers in sufficient quantities for biophysical, cellular, and in vivo studies. To address these challenges, we developed new protocols to generate different stable forms of unmodified and small-molecule-induced TDP-43 oligomers. Our results showed that co-incubation of TDP-43 with small molecules, such as epigallocatechin gallate (EGCG), dopamine, and 4-hydroxynonenal (4-HNE), increased the production yield of TDP-43 stable oligomers, which could be purified by size-exclusion chromatography. Interestingly, despite significant differences in the morphology and size distribution of the TDP-43 oligomer preparations revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS), they all retained the ability to bind to nucleotide DNA. Besides, circular dichroism (CD) analysis of these oligomers did not show much difference in the secondary structure composition. Surprisingly, none of these oligomer preparations could seed the aggregation of TDP-43 core peptide 279-360. Finally, we showed that all four types of TDP-43 oligomers exert very mild cytotoxicity to primary neurons. Collectively, our results suggest that functional TDP-43 oligomers can be selectively stabilized by small-molecule compounds. This strategy may offer a new approach to halt TDP-43 aggregation in various proteinopathies.

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.

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.

The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections.

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.