Α-syn Oligomers Research Articles

Novel Insights into Parkin-Mediated Mitochondrial Dysfunction and "Mito-Inflammation" in α-Synuclein Toxicity. The Role of the cGAS-STING Signalling Pathway.

The prevalence of age-related neurodegenerative diseases, such as Parkinson's disease (PD) and related disorders continues to grow worldwide. Increasing evidence links intracellular inclusions of misfolded alpha-synuclein (α-syn) aggregates, so-called Lewy bodies (LB) and Lewy neuritis, to the progressive pathology of PD and other synucleinopathies. Our previous findings established that α-syn oligomers induce S-nitrosylation and deregulation of the E3-ubiquitin ligase Parkin, leading to mitochondrial disturbances in neuronal cells. The accumulation of damaged mitochondria as a consequence, together with the release of mitochondrial-derived damage-associated molecular patterns (mtDAMPs) could activate the innate immune response and induce neuroinflammation ("mito-inflammation"), eventually accelerating neurodegeneration. However, the molecular pathways that transmit pro-inflammatory signals from damaged mitochondria are not well understood. One of the proposed pathways could be the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) (cGAS-STING) pathway, which plays a pivotal role in modulating the innate immune response. It has recently been suggested that cGAS-STING deregulation may contribute to the development of various pathological conditions. Especially, its excessive engagement may lead to neuroinflammation and appear to be essential for the development of neurodegenerative brain diseases, including PD. However, the precise molecular mechanisms underlying cGAS-STING pathway activation in PD and other synucleinopathies are not fully understood. This review focuses on linking mitochondrial dysfunction to neuroinflammation in these disorders, particularly emphasizing the role of the cGAS-STING signaling. We propose the cGAS-STING pathway as a critical driver of inflammation in α-syn-dependent neurodegeneration and hypothesize that cGAS-STING-driven "mito-inflammation" may be one of the key mechanisms promoting the neurodegeneration in PD. Understanding the molecular mechanisms of α-syn-induced cGAS-STING-associated "mito-inflammation" in PD and related synucleinopathies may contribute to the identification of new targets for the treatment of these disorders.

Alpha-Synuclein Oligomers Driven T1-T2 Switchable Nanoprobes for Early and Accurate Diagnosis of Parkinson's Disease.

The alpha-synuclein (α-syn) oligomers hold a central role in the pathology of Parkinson's disease (PD). Achieving accurate detection of α-syn oligomers in vivo presents a promising avenue for early and accurate diagnosis of PD. Magnetic resonance imaging (MRI), with non-invasion and exceptional tissue penetration, offers a potent tool for visualizing α-syn oligomers in vivo. Nonetheless, ensuring diagnostic specificity remains a formidable challenge. Herein, a novel MRI probe (ASOSN) is introduced, which encompasses highly sensitive antiferromagnetic nanoparticles functionalized with single-chain fragment variable antibodies, endowing it with the capacity for discerning recognition and binding to α-syn oligomers and triggering a switchable T1-T2 MRI signal. Significantly, ASOSN possesses the unique capability to accurately discriminate α-syn oligomers from neuroinflammation in vivo. Moreover, ASOSN facilitates the non-invasive and precise visualizing of endogenous α-syn oligomers in living systems. This innovative design heralds the development of a non-invasive visualization strategy for α-syn oligomers, marking a pivotal advancement for early and accurate diagnosis of PD.

FC1: Effect of Transcranial Direct Current Stimulation (tDCS) on Left Dorsolateral Prefrontal Cortex (DLPFC) in Dementia with Lewy Bodies (DLB)

Introduction:tDCS application to the DLPFC is associated with the improvements of executive function, memory enhancement, language, processing speed, global cognitive symptoms and apathy over time after treatment. DLB is the second most common form of degenerative dementia. There is no FDA-approved medications that can slow, stop or improve the progression of cognitive declines in DLB. Identifying effective treatments is a critical issue for DLB. In neuropathology, extracelluar α-syn oligomers interfere with the expression of long-term potentiation(LTP), and influence memory and learning. tDCS has been proposed to affect long-term synaptic plasticity through LTP and long-term depression, thereby improving cognitive ability. So far, only two studies have evaluated the effect of tDCS in DLB. In this pilot study, we investigate the effect of tDCS on left DLPFC in DLB.Method:Fourteen DLB aged 55-90 years (mean age 76.4, with 4 males and 10 females) were included in a double-blind, randomized, sham-controlled cross over design study. DLB diagnostics is according to DSM-5 criteria. CDR ratings for DLB participants ranged from 0.5 to 2. The active tDCS (or sham) process consists of daily sessions of active tDCS (or sham) for 10 consecutive days. The anodal electrode was placed over the left DLPFC and the cathodal electrode was placed over the right supraorbital area, with a current intensity of 2 mA and an electrode size of 25 cm2 for 30 min in a session. Before and after these treatment sessions, all subjects received a series of neuropsychological tests, including CDR, MMSE, CASI, NPI and WCST. Chi-square test, Wilcoxon signed ranks test and Mann-Whitney U test were used to assess differences in participant demographic characteristics and to compare differences among groups.Results:The active tDCS group showed significant improvements on the three items of CASI, ‘language ability’, ‘concentration and calculation’, ‘categorical verbal fluency’, after active stimulations. There is no improvement in MMSE, CASI, NPI and WCST scores in the sham groups.Conclusion:These results suggest that left DLPFC anodal, and right deltoid cathodal tDCS, may have some cognitive benefits in DLB. Larger-scale trials are needed to confirm the effect of tDCS in DLB.Key words: Transcranial Direct Current Stimulation, Dementia with Lewy Bodies, cognitive function, Wisconsin Card Sorting Test, left DLPFC

Non-invasive systemic viral delivery of human alpha-synuclein mimics selective and progressive neuropathology of Parkinson’s disease in rodent brains

BackgroundAlpha-synuclein (α-syn) aggregation into proteinaceous intraneuronal inclusions, called Lewy bodies (LBs), is the neuropathological hallmark of Parkinson’s disease (PD) and related synucleinopathies. However, the exact role of α-syn inclusions in PD pathogenesis remains elusive. This lack of knowledge is mainly due to the absence of optimal α-syn-based animal models that recapitulate the different stages of neurodegeneration.MethodsHere we describe a novel approach for a systemic delivery of viral particles carrying human α-syn allowing for a large-scale overexpression of this protein in the mouse brain. This approach is based on the use of a new generation of adeno-associated virus (AAV), AAV-PHP.eB, with an increased capacity to cross the blood-brain barrier, thus offering a viable tool for a non-invasive and large-scale gene delivery in the central nervous system.ResultsUsing this model, we report that widespread overexpression of human α-syn induced selective degeneration of dopaminergic (DA) neurons, an exacerbated neuroinflammatory response in the substantia nigra and a progressive manifestation of PD-like motor impairments. Interestingly, biochemical analysis revealed the presence of insoluble α-syn oligomers in the midbrain. Together, our data demonstrate that a single non-invasive systemic delivery of viral particles overexpressing α-syn prompted selective and progressive neuropathology resembling the early stages of PD.ConclusionsOur new in vivo model represents a valuable tool to study the role of α-syn in PD pathogenesis and in the selective vulnerability of nigral DA neurons; and offers the opportunity to test new strategies targeting α-syn toxicity for the development of disease-modifying therapies for PD and related disorders.

The potential of Rhein's aromatic amines for Parkinson's disease prevention and treatment: α-Synuclein aggregation inhibition and disaggregation of preformed fibers

The aggregation of α-Syn is a pivotal mechanism in Parkinson's disease (PD). Effectively maintaining α-Syn proteostasis involves both inhibiting its aggregation and promoting disaggregation. In this study, we developed a series of aromatic amide derivatives based on Rhein. Two of these compounds, 4,5-dihydroxy-N-(3-hydroxyphenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a5) and 4,5-dihydroxy-N-(2-hydroxy-4-chlorophenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a8), exhibited good binding affinities to α-Syn residues, demonstrating promising inhibitory activity against α-Syn aggregation in vitro, with low IC50 values (1.35 and 1.08 μM, respectivly). These inhibitors acted throughout the entire aggregation process by stabilizing α-Syn's conformation and preventing the formation of β-sheet aggregates. They also effectively disassembled preformed α-Syn oligomers and fibrils. Preliminary mechanistic insights indicated that they bound to the specific domain within fibrils, inducing fibril instability, collapse, and the formation of smaller aggregates and monomeric α-Syn units. This research underscores the therapeutic potential of Rhein's aromatic amides in targeting α-Syn aggregation for PD treatment and suggests broader applications in managing and preventing neurodegenerative diseases.

Recombinant human HspB5-ACD structural domain inhibits neurotoxicity by regulating pathological α-Syn aggregation

The treatment of Parkinson's disease is a global medical challenge. α-Synuclein (α-Syn) is the causative protein in Parkinson's disease and is closely linked to its progression. Therefore, inhibiting the pathological aggregation of α-Syn and its neurotoxicity is essential for the treatment of Parkinson's disease. In this study, α-Syn and recombinant human HspB5-ACD structural domain protein (AHspB5) were produced using the BL21(DE3) E. coli prokaryotic expression system, and then the role and mechanism of AHspB5 in inhibiting the pathological aggregation of α-Syn and its neurotoxicity were investigated. As a result, we expressed α-Syn and AHspB5 proteins and characterised the proteins. In vitro experiments showed that AHspB5 could inhibit the formation of α-Syn oligomers and fibrils; in cellular experiments, AHspB5 could prevent α-Syn-induced neuronal cell dysfunction, oxidative stress damage and apoptosis, and its mechanism of action was related to the TH-DA pathway and mitochondria-dependent apoptotic pathway; in animal experiments, AHspB5 could inhibit behavioural abnormalities, oxidative stress damage and loss of dopaminergic neurons. In conclusion, this work is expected to elucidate the mechanism and biological effects of AHspB5 on the pathological aggregation of α-Syn, providing a new pathway for the treatment of Parkinson's disease and laying the foundation for recombinant AHspB5.

4-Arylidene curcumin derivatives in vitro inhibit α-Synuclein aggregation and disaggregate the preformed fibril

This study focuses on the misfolding and aggregation of α-Syn as a central mechanism linking various pathological processes in PD. Maintaining α-Syn proteostasis through suitable inhibitors emerges as an effective approach to prevent PD. A more efficient strategy for PD treatment involves disintegrating neurotoxic oligomers and fibrils into normal functional α-Syn using inhibitors. To this end, a series of 4-arylidene curcumin derivatives were synthesized with a sheet-like conjugated skeleton and higher binding energies with α-Syn residues. Among these derivatives, three candidate compounds exhibited promising α-Syn aggregation inhibitory activities in vitro, with IC50 values as low as 0.61 μM. The inhibitory action extended throughout the entire aggregation process, stabilizing α-Syn proteostasis conformation and preventing β-sheets aggregation. Furthermore, the candidate compounds demonstrated effective disintegration capabilities against preformed α-Syn oligomers and fibrils. Initial mechanistic investigations indicated that the inhibitors may bind to a specific domain within the fibril, inducing fibril instability and subsequent collapse. This process resulted in the formation of a complex system of aggregates with smaller sizes and monomers. Overall, these findings provide valuable insights into the potential of 4-arylidene curcumin derivatives as therapeutic agents for targeting α-Syn aggregation in PD treatment.

Distinct molecular mechanisms contribute to the reduction of melanoma growth and tumor pain after systemic and local depletion of alpha-Synuclein in mice.

Epidemiological studies show a coincidence between Parkinson's disease (PD) and malignant melanoma. It has been suggested that this relationship is due, at least in part, to modulation of alpha-Synuclein (αSyn/Snca). αSyn oligomers accumulate in PD, which triggers typical PD symptoms, and in malignant melanoma, which increases the proliferation of tumor cells. In addition, αSyn contributes to non-motor symptoms of PD, including pain. In this study, we investigated the role of αSyn in melanoma growth and melanoma-induced pain in a mouse model using systemic and local depletion of αSyn. B16BL6 wild-type as well as αSyn knock-down melanoma cells were inoculated into the paws of αSyn knock-out mice and wild-type mice, respectively. Tumor growth and tumor-induced pain hypersensitivity were assessed over a period of 21 days. Molecular mechanisms were analyzed by RT-PCR and Western Blot in tumors, spinal cord, and sciatic nerve. Our results indicate that both global and local ablation of Snca contribute to reduced tumor growth and to a reduction of tumor-induced mechanical allodynia, though mechanisms contributing to these effects differ. While injection of wild-type cells in Snca knock-out mice strongly increased the immune response in the tumor, local Snca knock-down decreased autophagy mechanisms and the inflammatory reaction in the tumor. In conclusion, a knockdown of αSyn might constitute a promising approach to inhibiting the progression of melanoma and reducing tumor-induced pain.

Conformational dynamics of α-synuclein and study of its intramolecular forces in the presence of selected compounds

Protein misfolding and aggregation play crucial roles in amyloidogenic diseases through the self-assembly of intrinsically disordered proteins (IDPs) in type II diabetes (T2D), Alzheimer's disease (AD) and Parkinson’s disease (PD). PD is the most common neurodegenerative disorder after AD, and is associated with the loss of dopaminergic signaling, which causes motor and nonmotor signs and symptoms. Lewy bodies and Lewy neurites are common pathological hallmarks of PD that are mainly composed of aggregates of disordered α-synuclein (α-Syn). There have been many efforts to develop chemical compounds to prevent aggregation or facilitate disruption of the aggregates. Furthermore, the roles and interactions of many compounds have yet to be revealed at the atomistic level, especially their impacts on the dynamics and chain-chain interactions of the oligomers, which are of interest in this study. The conformational diversity and detailed interactions among homo-oligomer chains of α-Syn are not fully discovered; identifying these might help uncover a practical approach to developing a potent therapy. In this study, we used an in-silico investigation to address the conformational diversity of α-Syn oligomer. The roles of several point mutations in protein aggregation in PD are known; we take this further by evaluating the interaction energies and contributions of all residues in stability and residue-chain interactions. In this study, we docked chemical derivatives of three compounds with high drug-likeness properties to evaluate the roles of our ligands in the conformational dynamicity of the oligomers, with emphasis on intramolecular forces. Free energy evaluation of the modeled inter and intramolecular interactions through MD simulation shows effective interaction and binding between α-Syn and our compounds. However, we find that they do not significantly disrupt the chain-chain interactions, compared to unliganded simulation.

Research Progress of α-Synuclein Aggregation Inhibitors for Potential Parkinson's Disease Treatment.

Parkinson's disease (PD) is characterized by fibrillation of disordered proteins known as Lewy bodies in the substantia nigra that also undergo progressive neurodegeneration. The aggregation of α-synuclein (α-syn) is a hallmark and potentially a critical step in the development of Parkinson's disease and other synucleinopathies. The synaptic vesicle protein α-syn is a small, abundant, highly conserved disordered protein and the causative agent of neurodegenerative diseases. Several novel pharmacologically active compounds are used to treat PD and other neurodegenerative disorders. Though, the mechanism through which these molecules inhibit the α-syn aggregation is still not fully understood. This review article is focused on the recent advancements in compounds that can inhibit the development of α-syn fibrillation and oligomerization. The current review article is based on the most recent and frequently cited papers from Google Scholar, SciFinder, and Researchgate sources. In the progression of PD, the mechanism of α-syn aggregation involves the structural transformation from monomers into amyloid fibrils. As the accumulation of α-syn in the brain has been linked to many disorders, the recent search for disease-modifying medications mainly focused on modifying the α-syn aggregation. This review contains a detailed report of literature findings and illustrates the unique structural features, structure-activity relationship, and therapeutic potential of the natural flavonoids in the inhibition of α-syn are also discussed. Recently, many naturally occurring molecules such as curcumin, polyphenols, nicotine, EGCG, and stilbene have been recognized to inhibit the fibrillation and toxicity of α-syn. Therefore, knowing the α-synuclein filament's structure and how they originate will help invent particular biomarkers for synucleinopathies and develop reliable and effective mechanism-based therapeutics. We hope the information this review provides may help evaluate novel chemical compounds, such as α-syn aggregation inhibitors, and will contribute to developing novel drugs for treating Parkinson's disease.

The effects of post-translational modifications on alpha-synuclein aggregation and immune cell activation in Parkinson’s disease

Post-translational modifications (PTMs) of alpha-synuclein (α-syn) can alter protein aggregation propensity to affect α-syn oligomer and fibril formation. The inflammatory response in Parkinson’s disease (PD) is mediated by microglia, astrocytes, T cells, B cells, macrophages, and neutrophils, which respond to α-syn aggregates in an attempt to clear synucleinopathy and restore brain homeostasis. This review focuses on the effects of PTMs on α-syn aggregation and cell-specific immune responses to α-syn aggregates in the context of PD.

Elucidating the Role of Lipids in the Aggregation of Amyloidogenic Proteins.

The abrupt aggregation of misfolded proteins is linked to the onset and spread of amyloidogenic diseases, including diabetes type 2, systemic amyloidosis, and Alzheimer's (AD) and Parkinson's diseases (PD). Although the exact cause of these pathological processes is unknown, a growing body of evidence suggests that amyloid diseases are triggered by misfolded or unfolded proteins, forming highly toxic oligomers. These transient species exhibit high structural and morphological heterogeneity. Protein oligomers can also propagate into β-sheet-rich filaments that braid and coil with other filaments to form amyloid fibrils and supramolecular structures with both flat and twisted morphologies. Microscopic examination of protein deposits formed in the brains of both AD and PD patients revealed the presence of fragments of lipid membranes. Furthermore, nanoscale infrared analysis of ex vivo extracted fibrils revealed the presence of lipids in their structure (Zhaliazka, K.; Kurouski, D. Protein Sci. 2023, 32, e4598). These findings demonstrated that lipid bilayers could play an important role in the aggregation of misfolded proteins.Experimental findings summarized in this Account show that (i) lipids uniquely change the aggregation rate of amyloidogenic proteins. In this case, the observed changes in the rates directly depend on the net charge of the lipid and the length and saturation of lipid fatty acids (FAs). For instance, zwitterionic phosphatidylcholine (PC) with 14:0 FAs inhibited the aggregation of insulin, lysozyme, and α-synuclein (α-Syn), whereas anionic phosphatidylserine with the same FAs dramatically accelerated the aggregation rate of these proteins (Dou, T., et al. J. Phys. Chem. Lett. 2021, 12, 4407. Matveyenka, M., et al. FASEB J. 2022, 36, e22543. Rizevsky, S., et al. J. Phys. Chem. Lett. 2022, 13, 2467). Furthermore, (ii) lipids uniquely alter the secondary structure and morphology of protein oligomers and fibrils formed in their presence. Utilization of nano-infrared spectroscopy revealed that such aggregates, as well as ex vivo extracted fibrils, possessed lipids in their structure. These findings are significant because (iii) lipids uniquely alter the toxicity of amyloid oligomers and fibrils formed in their presence. Specifically, PC lowered the toxicity of insulin and lysozyme oligomers, whereas α-Syn oligomers formed in the presence of this phospholipid were found to be significantly more toxic to rat dopaminergic cells compared to α-Syn oligomers grown in the lipid-free environment. Thus, the toxicity of protein oligomers and fibrils is directly determined by the chemical structure of the lipid and the secondary structure of amyloidogenic proteins (Dou, T., et al. J. Phys. Chem. Lett. 2021, 12, 4407. Matveyenka, M., et al. FASEB J. 2022, 36, e22543. Rizevsky, S., et al. J. Phys. Chem. Lett. 2022, 13, 2467). Experimental results discussed in this Account also suggest that amyloidogenic diseases could be caused by pathological changes in the lipid composition of both plasma and organelle membranes, which, in turn, may trigger protein aggregation that results in the formation of highly toxic oligomers and fibrils. Finally, the Account discusses the effects of polyunsaturated FAs on the aggregation properties of amyloidogenic proteins. Experimental findings reported by the author's laboratory revealed that polyunsaturated FAs drastically accelerated the aggregation rate of both insulin and α-Syn as well as strongly changed the secondary structure of amyloid fibrils formed in their presence.

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-β (Aβ) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aβ plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aβ-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aβ-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

4-Oxo-2-nonenal-Induced α-Synuclein Oligomers Interact with Membranes in the Cell, Leading to Mitochondrial Fragmentation.

Oxidative stress and formation of cytotoxic oligomers by the natively unfolded protein α-synuclein (α-syn) are both connected to the development of Parkinson's disease. This effect has been linked to lipid peroxidation and membrane disruption, but the specific mechanisms behind these phenomena remain unclear. To address this, we have prepared α-syn oligomers (αSOs) in vitro in the presence of the lipid peroxidation product 4-oxo-2-nonenal and investigated their interaction with live cells using in-cell NMR as well as stimulated emission depletion (STED) super-resolution and confocal microscopy. We find that the αSOs interact strongly with organellar components, leading to strong immobilization of the protein's otherwise flexible C-terminus. STED microscopy reveals that the oligomers localize to small circular structures inside the cell, while confocal microscopy shows mitochondrial fragmentation and association with both late endosome and retromer complex before the SOs interact with mitochondria. Our study provides direct evidence for close contact between cytotoxic α-syn aggregates and membraneous compartments in the cell.

Genome-wide CRISPR-Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers.

Misfolding and aggregation of α-Synuclein (α-Syn), which are hallmark pathological features of neurodegenerative diseases such as Parkinson's disease (PD) and dementia with Lewy Bodies, continue to be significant areas of research. Among the diverse forms of α-Syn - monomer, oligomer, and fibril, the oligomer is considered the most toxic. However, the mechanisms governing α-Syn oligomerization are not yet fully understood. In this study, we utilized genome-wide CRISPR/Cas9 loss-of-function screening in human HEK293 cells to identify negative regulators of α-Syn oligomerization. We found that tetraspanin 3 (TSPAN3), a presumptive four-pass transmembrane protein, but not its homolog TSPAN7, significantly modulates α-Syn oligomer levels. TSPAN3 was observed to interact with α-Syn oligomers, regulate the amount of α-Syn oligomers on the cell membrane, and promote their degradation via the clathrin-AP2 mediated endo-lysosome pathway. Our findings highlight TSPAN3 as a potential regulator of α-Syn oligomers, presenting a promising target for future PD prevention and treatment strategies.

Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution.

Soluble oligomers of amyloid-forming proteins are implicated as toxic species in the context of several neurodegenerative diseases. Since the size and shape of these oligomers influence their toxicity, their biophysical characterization is essential for a better understanding of the structure-toxicity relationship. Amyloid oligomers are difficult to characterize by conventional approaches due to their heterogeneity in size and shape, their dynamic aggregation process, and their low abundance. This work demonstrates that resistive pulse measurements using polymer-coated solid-state nanopores enable single-particle-level characterization of the size and shape of individual αSyn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals good agreement with superior resolution by nanopore-based characterization. Moreover, nanopore-based analysis has the capability to combine rapid size analysis with an approximation of the oligomer shape. Applying this shape approximation to putatively toxic oligomeric species that range in size from 18 ± 7 aggregated monomers (10S) to 29 ± 10 aggregated monomers (15S) and in concentration from picomolar to nanomolar revealed oligomer shapes that agree well with previous estimates by cryo-EM with the added advantage that nanopore-based analysis occurs rapidly, in solution, and has the potential to become a widely accessible technique.

Unveiling How Hydroxytyrosol Destabilizes α-Syn Oligomers Using Molecular Simulations.

The etiology of Parkinson's disease (PD) is mainly linked to the α-synuclein (α-Syn) fibrillogenesis. Hydroxytyrosol (HT), also known as 3,4-dihydroxyphenylethanol, is a naturally occurring polyphenol, found in extra virgin olive oil, and has been shown to have cardioprotective, anticancer, antiobesity, and antidiabetic properties. HT has neuroprotective benefits in neurodegenerative diseases and lessens the severity of PD by reducing the aggregation of α-Syn and destabilizing the preformed toxic α-Syn oligomers. However, the molecular mechanism by which HT destabilizes α-Syn oligomers and alleviates the accompanying cytotoxicity remains unexplored. The impact of HT on the α-Syn oligomer structure and its potential binding mechanism was examined in this work by employing molecular dynamics (MD) simulations. The secondary structure analysis depicted that HT significantly reduces the β-sheet and concomitantly increases the coil content of α-Syn trimer. Visualization of representative conformations from the clustering analysis depicted the hydrogen bond interactions of the hydroxyl groups in HT with the N-terminal and nonamyloid-β component (NAC) region residues of α-Syn trimer, which, in turn, leads to the weakening of interchain interactions in α-Syn trimer and resulted in the disruption of the α-Syn oligomer. The binding free energy calculations depict that HT binds favorably to α-Syn trimer (ΔGbinding = -23.25 ± 7.86 kcal/mol) and a notable reduction in the interchain binding affinity of α-Syn trimer on the incorporation of HT, which, in turn, highlights its potential to disrupt α-Syn oligomers. The current research provided mechanistic insights into the destabilization of α-Syn trimer by HT, which, in turn, will provide new clues for developing therapeutics against PD.

5-Nitro-1,2-benzothiazol-3-amine and N-Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α-Synuclein and Tau Aggregation.

Protein misfolding results in a plethora of known diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, transthyretin-related amyloidosis, type 2 diabetes, Lewy body dementia, and spongiform encephalopathy. To provide a diverse portfolio of therapeutic small molecules with the ability to reduce protein misfolding, we evaluated a set of 13 compounds: 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives containing urea (1), thiourea (2), sulfonamide (3), triazole (4), and triazine (5) linker. In addition, we explored small modifications on a very potent antioligomer 5-nitro-1,2-benzothiazol-3-amine (5-NBA) (compounds 6-13). This study aims to define the activity of BTA and its derivatives on a variety of prone-to-aggregate proteins such as transthyretin (TTR81-127, TTR101-125), α-synuclein (α-syn), and tau isoform 2N4R (tau 2N4R) through various biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation of the previously mentioned proteins after treatment with BTA and its derivatives. Antifibrillary activity was confirmed using transmission electron microscopy (TEM). Photoreactive cross-linking assay (PICUP) was utilized to detect antioligomer activity and lead to the identification of 5-NBA (at low micromolar concentration) and compound 13 (at high concentration) as the most promising in reducing oligomerization. 5-NBA and not BTA inhibited the inclusion formation based on the cell-based assay using M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. 5-NBA abrogated the fibril, oligomer, and inclusion formation in a dose-dependent manner. 5-NBA derivatives could be the key to mitigate protein aggregation. In the future, the results made from this study will provide an initial platform to generate more potent inhibitors of α-syn and tau 2N4R oligomer and fibril formation.

Oligomeric Alpha-Synuclein and STX-1A from Neural-Derived Extracellular Vesicles (NDEVs) as Possible Biomarkers of REM Sleep Behavior Disorder in Parkinson's Disease: A Preliminary Cohort Study.

REM sleep behavior disorder (RBD) has a tighter link with synucleinopathies than other neurodegenerative disorders. Parkinson's Disease (PD) patients with RBD have a more severe motor and cognitive impairment; biomarkers for RBD are currently unavailable. Synaptic accumulation of α-Syn oligomers and their interaction with SNARE proteins is responsible for synaptic dysfunction in PD. We verified whether oligomeric α-Syn and SNARE components in neural-derived extracellular vesicles (NDEVs) in serum could be biomarkers for RBD. Forty-seven PD patients were enrolled, and the RBD Screening Questionnaire (RBDSQ) was compiled. A cut-off score > 6 to define probable RBD (p-RBD) and probable non-RBD (p non-RBD) was used. NDEVs were isolated from serum by immunocapture, and oligomeric α-Syn and SNARE complex components VAMP-2 and STX-1 were measured by ELISA. NDEVs' STX-1A resulted in being decreased in p-RBD compared to p non-RBD PD patients. A positive correlation between NDEVs' oligomeric α-Syn and RBDSQ total score was found (p = 0.032). Regression analysis confirmed a significant association between NDEVs' oligomeric α-Syn concentration and RBD symptoms (p = 0.033) independent from age, disease duration, and motor impairment severity. Our findings suggest that synuclein-mediated neurodegeneration in PD-RBD is more diffuse. NDEVs' oligomeric α-Syn and SNARE complex components' serum concentrations could be regarded as reliable biomarkers for the RBD-specific PD endophenotype.

Dopamine-Induced Oligomers of α-Synuclein Inhibit Amyloid Fibril Growth and Show No Toxicity.

Parkinson's disease is characterized by the selective death of dopaminergic neurons in the midbrain and accumulation of amyloid fibrils composed of α-synuclein (αSyn). Current treatment involves approaches that compensate the death of dopaminergic neurons by increasing the dopamine levels in remaining cells. However, dopamine can interact with αSyn and produce oligomeric species which were reported to be toxic in many models. We studied formation of dopamine-induced αSyn oligomers and their effect on the αSyn aggregation. Using the Thioflavin T kinetic assay, we have shown that small oligomers efficiently inhibit αSyn fibrillization by binding to fibril ends and blocking the elongation. Moreover, all the fractions of oligomer species proved to be nontoxic in the differentiated SH-SY5Y cell model and showed negligible neurotoxicity on isolated rat synaptosomes. The observed inhibition is an important insight in understanding of dopamine-enhancing therapy on Parkinson's disease progression and explains the absence of pathology enhancement.