Thi Hong Van Nguyen

The aggregation of α-synuclein (αSyn), a 140-mer protein, has been implicated in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. KTKEGV pseudo-repeats (KRs) in the sequence of αSyn are key mediators of its prion-like propagation and neurodegeneration. Despite the availability of symptomatic treatments, no current therapy effectively delays disease progression. Here we report a 77-nucleotide RNA aptamer called 1R6, obtained through in vitro selection, that binds with high affinity and selectivity to αSyn1-95. 1R6 significantly inhibits αSyn oligomerization and β-sheet-rich fibril assembly and promotes disaggregation of preformed fibrils. Additionally, 1R6 suppresses αSyn seeding, as determined by a FRET-based biosensor-cell assay. Cellular studies reveal that 1R6 co-transfection completely prevents αSyn-induced cytotoxicity. To assess the protective effects of 1R6 in vivo, we use a Drosophila melanogaster model expressing human αSyn in neurons. Flies fed 1R6 show improved locomotor activity, reduced photoreceptor degeneration, and decreased αSyn levels in the head. Structural characterization using ¹H-¹⁵N heteronuclear multiple quantum correlation nuclear magnetic resonance experiments demonstrates that 1R6 targets KR motifs, a finding further supported by in silico simulations. Our findings support further development of RNA aptamers, such as 1R6, including evaluation of efficacy, stability, delivery, and immune responses in mammalian systems, highlighting their potential as therapeutic candidates for synucleinopathies.

Trichalcogenides with linearly catenated chalcogen atoms (S, Se, and Te), exemplified by glutathione trisulfide, are increasingly recognized as key regulators of biological redox processes. However, their robust structural characterization remains limited because the NMR-active sulfur isotope (³³S, I = 3/2) is low-abundance and quadrupolar. The examination of chalcogen connection in heterologous trichalcogenides remains highly challenging. Recent studies have examined ⁷⁷Se and ¹²⁵Te as replacements for sulfur and applied ⁷⁷Se/¹²⁵Te NMR in biomolecules. In this study, we report the in situ synthesis of heterologous trichalcogenides of glutathione disulfide (GS-SG) by inserting a single chalcogen atom into a disulfide bond using chalcogen donors in a conventional NMR tube. The NMR-active isotopes ⁷⁷Se and ¹²⁵Te (I = 1/2) provide broad chemical-shift ranges and high sensitivity to structural and dynamic changes, enabling multinuclear NMR. ¹H DOSY and quantitative ¹H NMR confirm a single major species (>95% by NMR) and ¹H-detected ⁷⁷Se/¹²⁵Te heteronuclear multiple-bond correlation experiments demonstrate long-range coherence transfers. Heavy chalcogen insertion causes greater structural perturbation, making Se more suitable than Te. Density functional theory calculation revealed a smaller highest occupied molecular orbital–lowest unoccupied molecular orbital gap for GS-Te-SG, indicating its lower thermodynamic stability than GS-Se-SG. Consistent results were observed in the cystine-based experiments. To the best of our knowledge, for the first time, this study demonstrated that Se or Te can be inserted into the central site of disulfide bonding. In the biological assays, glutathione derivatives incorporating heavier chalcogens exhibit antiradical activities. This in situ synthetic and analytical framework expands the chemical space of biologically relevant chalcogenides.

The aggregation of α-synuclein (αSyn), a 140-mer protein, has been implicated in the pathogenesis of Parkinson′s disease, multiple system atrophy, and dementia with Lewy bodies. KTKEGV repeats (KR) of αSyn are key mediators of prion-like propagation and neurodegeneration. Despite the availability of symptomatic treatments, no current therapy effectively delays disease progression. Here we report a 77-nucleotide (nt) RNA aptamer (1R6) with potent affinity and selectivity for αSyn1-95 ( K D = 18 nM) through in vitro selection. 1R6 significantly inhibited αSyn oligomerization and β-sheet-rich fibril assembly and promoted disaggregation of preformed fibrils. Additionally, 1R6 suppressed αSyn seeding, as determined by FRET-based cellular biosensor cell assay. Cellular studies revealed that 1R6 cotransfection completely prevented αSyn-induced cytotoxicity. To assess the protective effects of 1R6 in vivo , we used a Drosophila melanogaster model expressing human αSyn in neurons. Flies fed with 1R6 showed improved locomotor defects, reduced photoreceptor degeneration, and decreased αSyn levels in the head. Structural characterization through ¹ H- ¹⁵ N heteronuclear multiple quantum correlation nuclear magnetic resonance experiments demonstrated that 1R6 targets KR motifs, a finding further supported by in silico simulations. Our findings indicate that RNA aptamers, such as 1R6, may represent promising therapeutic candidates for synucleinopathies, thus opening new avenues in the treatment of these diseases.

RNA-binding proteins (RBPs), key translation regulators, are thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). The pathological entities associated with ALS are known as “MetaAggregates”: heterogeneous coaggregates composed of amyloids, RBPs, and RNA G-quadruplexes (rG4s). In this study, to explore the molecular constituents of ALS-associated MetaAggregates, we developed a proteomic approach using a psoralen-conjugated RBP and crosslinked it with a biotinylated rG4 to enable the isolation of MetaAggregates from ALS brain extracts. Single-cell RNA-seq using in vitro ALS models identified ELAVL4 as a cytoplasmic RBP and revealed the enrichment of an IGFBP2-derived rG4 structure in ALS-specific neurons. Mass spectrometry and amyloidogenicity-based principal component analysis revealed 79 candidate proteins with roles in RNA processing, metabolism, trafficking, and stress responses. Docking simulations highlighted a subset of proteins with potential pro-aggregation characteristics, diverse cytosolic associations and functional links to RNA processing relevant to ALS. Through proteomic and in silico dissection of ALS-associated MetaAggregates, the findings of this study establish a conceptual framework for the exploration of unrecognized amyloidogenic drivers of neurodegeneration.

Human coronaviruses (HCoVs) include seven species: HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV-1, and SARS-CoV-2. The last three, classified as Betacoronaviruses, are highly transmissible and have caused severe pandemics. HCoV infections primarily affect the respiratory system, leading to symptoms such as dry cough, fever, and breath shortness, which can progress to acute respiratory failure and death. Beyond respiratory effects, increasing evidence links HCoVs to neurological dysfunction. However, distinguishing direct neural complications from preexisting disorders, particularly in the elderly, remains challenging. This study examines the association between HCoVs and neurodegenerative diseases like Alzheimer disease, Parkinson disease, Lewy body dementia, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease. It also presents the long-term neurological effects of HCoV infections and their differential impact across age groups and sexes. A key aspect of this study is the investigation of the sequence and structural similarities between amyloidogenic and HCoV spike proteins, which can provide insights into potential neuropathomechanisms.

Inhibition of amyloidogenic-protein oligomeriza- tion and aggregation is a promising therapy-development strategy for proteinopathies, such as Alzheimer’s and Parkinson’s diseases, in which proteins self-associate into a variety of abnormal, toxic assemblies. Despite discovery of numerous compounds modulating the self-assembly process in vitro, few have reached advanced clinical trials, and none have translated into effective therapy to date. A potential reason is a lack of clear mechanistic under- standing of the interaction between the inhibitors/modulators and the target metastable protein assemblies. A unique class of compounds targets specifically Lys residues, which have been shown to be important mediators of many amyloidogenic-protein aberrant self-assembly processes due to their participation in both electrostatic and hydrophobic interactions. Although seemingly paradoxical, as these compounds do not target a specific protein, compounds targeting Lys show a remarkable ability to selectively disrupt the interactions mediating abnormal protein self-assembly. Such compounds include covalent and noncovalent Lys-binding small molecules, as well as agents controlling Lys-post-translational modification (PTM). Recent advances in this area show that the application of Lys-targeting inhibitors in antiamyloid drug discovery campaigns and Lys-reactive rational-design approaches have led to intriguing results in multiple systems, including animal models of various proteinopathies. As this strategy is applicable and promising for targeting most of the proteins involved in proteinopathies, including amyloid β -protein, tau, and α -synuclein, here we highlight Lys-binding inhibitors of abnormal protein self-assembly leading to preclinical therapeutic applications for the central nervous system.

Arenaviruses are emerging enveloped negative-sense RNA viruses that cause neurological and hemorrhagic diseases in humans. Currently, no FDA-approved vaccine or therapeutic agent is available except for ribavirin, which must be administered early during infection for optimum efficacy. A hallmark of arenavirus infection is rapid and efficient immune suppression mediated by the exonuclease domain encoded by the nucleoprotein. This exonuclease is therefore an attractive target for the design of novel antiviral drugs since exonuclease inhibitors might not only have a direct effect on the enzyme but could also boost viral clearance through stimulation of the innate immune system of the host cell. Here,in silicoscreening and an enzymatic assay were used to identify a novel, specific but weak inhibitor of the arenavirus exonuclease, with IC₅₀values of 65.9 and 68.6 µMfor Mopeia virus and Lymphocytic choriomeningitis virus, respectively. This finding was further characterized using crystallographic and docking approaches. This study serves as a proof of concept and may have assigned a new therapeutic purpose for the bisphosphonate family, therefore paving the way for the development of inhibitors againstArenaviridae.

Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single-stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self-assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full-length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 +/- 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.

Arenaviridae is a family of viruses harbouring important emerging pathogens belonging to the Bunyavirales order. Like in other segmented negative strand RNA viruses, the nucleoprotein (NP) is a major actor of the viral life cycle being both (i) the necessary co-factor of the polymerase present in the L protein, and (ii) the last line of defence of the viral genome (vRNA) by physically hiding its presence in the cytoplasm. The NP is also one of the major players interfering with the immune system. Several structural studies of NP have shown that it features two domains: a globular RNA binding domain (NP-core) in its N-terminal and an exonuclease domain (ExoN) in its C-terminal. Further studies have observed that significant conformational changes are necessary for RNA encapsidation. In this review we revisited the most recent structural and functional data available on Arenaviridae NP, compared to other Bunyavirales nucleoproteins and explored the structural and functional implications. We review the variety of structural motif extensions involved in NP–NP binding mode. We also evaluate the major functional implications of NP interactome and the role of ExoN, thus making the NP a target of choice for future vaccine and antiviral therapy.