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.

High-throughput bioprinting of the nasal epithelium using patient-derived nasal epithelial cells.

Progenitor human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models through biofabrication. However, this approach has limitations in terms of achieving the intricate three-dimensional (3D) structure of the natural nasal epithelium. 3D bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of progenitor hNECs ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4 weeks air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes, such as disease modeling, immunological studies, and drug screening.

High-Throughput Bioprinting of the Nasal Epithelium using Patient-derived Nasal Epithelial Cells.

Human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models. However, the manual approach is slow, low-throughput and has limitations in terms of achieving the intricate 3D structure of the natural nasal epithelium in a uniform manner. 3D Bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of nasal progenitor cells ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4-week air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes such as disease modeling, immunological studies, and drug screening.

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.

Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors.

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

Snca-GFP Knock-In Mice Reflect Patterns of Endogenous Expression and Pathological Seeding.

α-Synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission but its misfolding is also strongly implicated in Parkinson's disease (PD) and other neurodegenerative synucleinopathies in which misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wild-type (wt) aSyn. We report here the development and characterization of a mouse line expressing an aSyn-green fluorescence protein (GFP) fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wt littermates. Importantly, similar to wt aSyn, aSyn-GFP disperses from synaptic vesicles on membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wt mice, albeit with attenuated kinetics because of the GFP-tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, thereby providing new insights into aSyn function in health and disease.