Α-syn Fibrils Research Articles

Tau accelerates α-synuclein aggregation and spreading in Parkinson’s disease

The aggregation and prion-like propagation of α-synuclein are involved in the pathogenesis of Parkinson's disease. However, the underlying mechanisms regulating the assembly and spreading of α-synuclein fibrils remain poorly understood. Tau co-deposits with α-synuclein in the brains of Parkinson's disease patients, suggesting a pathological interplay between them. Here we show that tau interacts with α-synuclein and accelerates its aggregation. Compared with pure α-synuclein fibrils, the tau-modified α-synuclein fibrils show enhanced seeding activity, inducing mitochondrial dysfunction, synaptic impairment and neurotoxicity in vitro. Injection of the tau-modified α-synuclein fibrils into the striatum of mice induces more severe α-synuclein pathology, motor dysfunction and cognitive impairment when compared with the mice injected with pure α-synuclein fibrils. Knockout of tau attenuates the propagation of α-synuclein pathology and Parkinson's disease-like symptoms both in mice injected with α-syn fibrils and α-syn A53T transgenic mice. In conclusion, tau facilitates α-synuclein aggregation and propagation in Parkinson's disease.

Hybrids of polyphenolic acids and xanthone, the potential preventive and therapeutic effects on PD: Design, synthesis, in vitro anti-aggregation of α-synuclein, and disaggregation against the existed α-synuclein oligomer and fibril

The misfolding and aggregation of α-Syn are the central mechanism linking and facilitating the other pathological mechanisms of PD. Maintaining α-Syn proteostasis by suitable inhibitors is an effective means to prevent PD. Disintegrating the neurotoxic oligomers and fibrils into the normal functional α-Syn by inhibitors is a more efficient way for PD treatment. This work synthesized two series hybrids of polyphenolic acids and xanthone. The hybrids possess a sheet-like conjugated skeleton and higher binding energies with α-Syn residues. Some compounds present well α-Syn aggregation inhibitory activities in vitro (IC50 down to 2.58 μM). The inhibitory action goes throughout the aggregation process from lag to the stationary phase by stabilizing α-Syn proteostasis conformation and preventing β-sheets aggregation. The candidate compounds with appropriate LogP values (2.02–3.11) present good disintegration abilities against the existed α-Syn oligomers and fibrils. The preliminary mechanism studies suggest that the inhibitors could quickly and randomly bind to the specific site closed to the β-sheet domain in the fibril, resulting in unstable and collapse of the protein fibril, yielding a complex system with aggregates of different sizes and monomers.

Ulvan inhibits α-synuclein fibrillation and disrupts the mature fibrils: In vitro and in vivo studies

Misfolding and aggregation of α-synuclein (α-syn) play a key role in the pathogenesis of Parkinson's disease (PD). Herein, the inhibitory effect of ulvan on α-syn fibrillogenesis was studied using thioflavin T fluorescence and atomic force microscope assays. It is shown that ulvan could inhibit α-syn fibrillogenesis in a dose-dependent manner. Based on the circular dichroism results, it is found that ulvan delays greatly the conformational transition from its initial random coil to β-sheet rich structure. The protective effect of ulvan against celllular death induced by α-syn aggregates was investigated by MTT colorimetric and cellular staining methods. It is found that ulvan protects greatly PC12 cells from α-syn fibril-induced cytotoxicity. In addition, ulvan disaggregates preformed α-syn fibrils and reduces cytotoxicity in a dose-dependent manner. Thereafter, the inhibitory effects of ulvan against α-syn fibrillogenesis were probed using Caenorhabditis elegans model NL5901 expressing human α-syn. It is found that ulvan extends the lifespan of NL5901 and recovers the lipid deposition by reducing the accumulation of α-syn. Finally, the molecular interactions between ulvan and α-syn pentamer was also explored using molecular docking. These findings suggest that ulvan can be pursued as a novel candidate drug for treatment of PD.

α-Synuclein Impacts on Intrinsic Neuronal Network Activity Through Reduced Levels of Cyclic AMP and Diminished Numbers of Active Presynaptic Terminals.

α-synuclein (α-Syn) is intimately linked to synucleinopathies like Parkinson’s disease and dementia with Lewy bodies. However, the pathophysiological mechanisms that are triggered by this protein are still largely enigmatic. α-Syn overabundance may cause neurodegeneration through protein accumulation and mitochondrial deterioration but may also result in pathomechanisms independent from neuronal cell death. One such proposed pathological mechanism is the influence of α-Syn on non-stimulated, intrinsic brain activity. This activity is responsible for more than 90% of the brain’s energyconsumption, and is thus thought to play an eminent role in basic brain functionality. Here we report that α-Syn substantially disrupts intrinsic neuronal network burst activity in a long-term neuronal cell culture model. Mechanistically, the impairment of network activity originates from reduced levels of cyclic AMP and cyclic AMP-mediated signaling as well as from diminished numbers of active presynaptic terminals. The profound reduction of network activity due to α-Syn was mediated only by intracellularly expressed α-Syn, but not by α-Syn that is naturally released by neurons. Conversely, extracellular pre-formed fibrils of α-Syn mimicked the effect of intracellular α-Syn, suggesting that they trigger an off-target mechanism that is not activated by naturally released α-Syn. A simulation-based model of the network activity in our cultures demonstrated that even subtle effect sizes in reducing outbound connectivity, i.e., loss of active synapses, can cause substantial global reductions in non-stimulated network activity. These results suggest that even low-level loss of synaptic output capabilities caused by α-Syn may result in significant functional impairments in terms of intrinsic neuronal network activity. Provided that our model holds true for the human brain, then α-Syn may cause significant functional lesions independent from neurodegeneration.

PAAN/MIF nuclease inhibition prevents neurodegeneration in Parkinson’s disease

Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication invivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.

Unique seeding profiles and prion-like propagation of synucleinopathies are highly dependent on the host in human α-synuclein transgenic mice.

α-synuclein (αSyn) is an intrinsically disordered protein which can undergo structural transformations, resulting in the formation of stable, insoluble fibrils. αSyn amyloid-type nucleation can be induced by misfolded 'seeds' serving as a conformational template, tantamount to the prion-like mechanism. Accumulation of αSyn inclusions is a key feature of dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), and are found as additional pathology in Alzheimer's disease (AD) such as AD with amygdala predominant Lewy bodies (AD/ALB). While these disorders accumulate the same pathological protein, they exhibit heterogeneity in clinical and histological features; however, the mechanism(s) underlying this variability remains elusive. Accruing data from human autopsy studies, animal inoculation modeling, and in vitro characterization experiments, have lent credence to the hypothesis that conformational polymorphism of the αSyn amyloid-type fibril structure results in distinct "strains" with categorical infectivity traits. Herein, we directly compare the seeding abilities and outcome of human brain lysates from these diseases, as well as recombinant preformed human αSyn fibrils by the intracerebral inoculation of transgenic mice overexpressing either human wild-type αSyn or human αSyn with the familial A53T mutation. Our study has revealed that the initiating inoculum heavily dictates the phenotypic and pathological course of disease. Interestingly, we have also established relevant host-dependent distinctions between propagation profiles, including burden and spread of inclusion pathology throughout the neuroaxis, as well as severity of neurological symptoms. These findings provide compelling evidence supporting the hypothesis that diverse prion-type conformers may explain the variability seen in synucleinopathies.

A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity.

The alpha-synuclein mutation E83Q, the first in the NAC domain of the protein, was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it markedly accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy body (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induces higher seeding activity, and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in brains of patients with Parkinson’s disease.

Neuropathology of α-synuclein in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive movement disability accompanied by non-motor symptoms. The neuropathology hallmark of PD is the loss of dopaminergic neurons predominantly in the substantia nigra pars compacta and the presence of intracellular inclusions termed Lewy bodies (LBs), which are mainly composed of α-synuclein (αSyn). Detailed staging based on the distribution and progression pattern of αSyn pathology in the postmortem brains of PD patients revealed correlation with the clinical phenotypes but not invariably. Cumulative evidence from cell and animal studies has implied that αSyn propagation contributes to the anatomical spread of αSyn pathology in the brain. Here, we recount the studies over the past two centuries on the anatomopathological foundations of PD documented. We also review studies on the structural analysis of αSyn and LBs, Braak staging of αSyn pathology, the cell-to-cell propagation of αSyn as well as αSyn fibril polymorphisms, which underlie the phenotypic differences in synucleinopathies.

α-Synuclein Fibrils as Penrose Machines: A Chameleon in the Gear.

In 1957, Lionel Penrose built the first man-made self-replicating mechanical device and illustrated its function in a series of machine prototypes, prefiguring our current view of the genesis and the proliferation of amyloid fibrils. He invented and demonstrated, with the help of his son Roger, the concepts that decades later, would become the fundamentals of prion and prion-like neurobiology: nucleation, seeding and conformational templating of monomers, linear polymer elongation, fragmentation, and spread. He published his premonitory discovery in a movie he publicly presented at only two conferences in 1958, a movie we thus reproduce here. By making a 30-year-jump in the early 90’s, we evoke the studies performed by Peter Lansbury and his group in which α-Synuclein (α-Syn) was for the first time (i) compared to a prion; (ii) shown to contain a fibrillization-prone domain capable of seeding its own assembly into fibrils; (iii) identified as an intrinsically disordered protein (IDP), and which, in the early 2000s, (iv) was described by one of us as a protein chameleon. We use these temporally distant breakthroughs to propose that the combination of the chameleon nature of α-Syn with the rigid gear of the Penrose machine is sufficient to account for a phenomenon that is of current interest: the emergence and the spread of a variety of α-Syn fibril strains in α-Synucleinopathies.

Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo.

Parkinson’s disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.

Neurons with Cat's Eyes: A Synthetic Strain of α-Synuclein Fibrils Seeding Neuronal Intranuclear Inclusions.

The distinct neuropathological features of the different α-Synucleinopathies, as well as the diversity of the α-Synuclein (α-Syn) intracellular inclusion bodies observed in post mortem brain sections, are thought to reflect the strain diversity characterizing invasive α-Syn amyloids. However, this “one strain, one disease” view is still hypothetical, and to date, a possible disease-specific contribution of non-amyloid factors has not been ruled out. In Multiple System Atrophy (MSA), the buildup of α-Syn inclusions in oligodendrocytes seems to result from the terminal storage of α-Syn amyloid aggregates first pre-assembled in neurons. This assembly occurs at the level of neuronal cytoplasmic inclusions, and even earlier, within neuronal intranuclear inclusions (NIIs). Intriguingly, α-Syn NIIs are never observed in α-Synucleinopathies other than MSA, suggesting that these inclusions originate (i) from the unique molecular properties of the α-Syn fibril strains encountered in this disease, or alternatively, (ii) from other factors specifically dysregulated in MSA and driving the intranuclear fibrillization of α-Syn. We report the isolation and structural characterization of a synthetic human α-Syn fibril strain uniquely capable of seeding α-Syn fibrillization inside the nuclear compartment. In primary mouse cortical neurons, this strain provokes the buildup of NIIs with a remarkable morphology reminiscent of cat’s eye marbles (see video abstract). These α-Syn inclusions form giant patterns made of one, two, or three lentiform beams that span the whole intranuclear volume, pushing apart the chromatin. The input fibrils are no longer detectable inside the NIIs, where they become dominated by the aggregation of endogenous α-Syn. In addition to its phosphorylation at S129, α-Syn forming the NIIs acquires an epitope antibody reactivity profile that indicates its organization into fibrils, and is associated with the classical markers of α-Syn pathology p62 and ubiquitin. NIIs are also observed in vivo after intracerebral injection of the fibril strain in mice. Our data thus show that the ability to seed NIIs is a strain property that is integrally encoded in the fibril supramolecular architecture. Upstream alterations of cellular mechanisms are not required. In contrast to the lentiform TDP-43 NIIs, which are observed in certain frontotemporal dementias and which are conditional upon GRN or VCP mutations, our data support the hypothesis that the presence of α-Syn NIIs in MSA is instead purely amyloid-strain-dependent.

In vivo imaging of alpha-synuclein with antibody-based PET

The protein alpha-synuclein (αSYN) plays a central role in synucleinopathies such as Parkinsons's disease (PD) and multiple system atrophy (MSA). Presently, there are no selective αSYN positron emission tomography (PET) radioligands that do not also show affinity to amyloid-beta (Aβ). We have previously shown that radiolabeled antibodies, engineered to enter the brain via the transferrin receptor (TfR), is a promising approach for PET imaging of intrabrain targets. In this study, we used this strategy to visualize αSYN in the living mouse brain. Five bispecific antibodies, binding to both the murine TfR and αSYN were generated and radiolabeled with iodine-125 or iodine-124. All bispecific antibodies bound to αSYN and mTfR before and after radiolabelling in an ELISA assay, and bound to brain sections prepared from αSYN overexpressing mice as well as human PD- and MSA subjects, but not control tissues in autoradiography. Brain concentrations of the bispecific antibodies were between 26 and 63 times higher than the unmodified IgG format 2 h post-injection, corresponding to about 1.5% of the injected dose per gram brain tissue. Additionally, intrastriatal αSYN fibrils were visualized with PET in an αSYN deposition mouse model with one of the bispecific antibodies, [124I]RmAbSynO2-scFv8D3. However, PET images acquired in αSYN transgenic mice with verified brain pathology injected with [124I]RmAbSynO2-scFv8D3 and [124I]RmAb48-scFv8D3 showed no increase in antibody retention compared to WT mice. Despite successful imaging of deposited extracellular αSYN using a brain-penetrating antibody-based radioligand with no cross-specificity towards Aβ, this proof-of-concept study demonstrates challenges in imaging intracellular αSYN inclusions present in synucleinopathies.

Interleukin-6 triggers toxic neuronal iron sequestration in response to pathological α-synuclein.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Lysophosphatidylcholine acyltransferase 1 promotes pathology and toxicity in two distinct cell-based alpha-synuclein models

Alpha-synuclein (αSyn) pathology is a hallmark of Parkinson’s disease. Here we show that lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a regulator of αSyn pathology and cytotoxicity. LPCAT1 is upregulated by αSyn E35K E46K E61K (3K) in human M17 neuroblastoma cells and primary rat cortical neurons, and in postmortem brain tissue from PD patients with confirmed αSyn aggregate pathology. Suppression of LPCAT1 reduces αSyn accumulations and toxicity in our neuroblastoma αSyn 3K overexpression model. Further overexpression of LPCAT1 promotes pS129 αSyn positive aggregation in primary neurons in the αSyn pre-formed fibril (PFF) model. A phospholipid product of LPCAT1 enzymatic activity, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, similarly promotes neuronal PFF seeded aggregation. Using a pH sensitive PFF model we provide evidence that αSyn fibrils have altered endo-lysosomal processing under LPCAT1 enhancement, suggesting less aggregate degradation. Our data demonstrates that LPCAT1 and associated phospholipids can regulate αSyn pathology.

Synapsin III gene silencing redeems alpha-synuclein transgenic mice from Parkinson's disease-like phenotype

Fibrillary aggregated α-synuclein (α-syn) deposition in Lewy bodies (LB) characterizes Parkinson's disease (PD) and is believed to trigger dopaminergic synaptic failure and a retrograde terminal-to-cell body neuronal degeneration. We described that the neuronal phosphoprotein synapsin III (Syn III) cooperates with α-syn to regulate dopamine (DA) release and can be found in the insoluble α-syn fibrils composing LB. Moreover, we showed that α-syn aggregates deposition, and the associated onset of synaptic deficits and neuronal degeneration occurring following adeno-associated viral vectors-mediated overexpression of human α-syn in the nigrostriatal system are hindered in Syn III knock out mice. This supports that Syn III facilitates α-syn aggregation. Here, in an interventional experimental design, we found that by inducing the gene silencing of Syn III in human α-syn transgenic mice at PD-like stage with advanced α-syn aggregation and overt striatal synaptic failure, we could lower α-syn aggregates and striatal fibers loss. In parallel, we observed recovery from synaptic vesicles clumping, DA release failure, and motor functions impairment. This supports that Syn III consolidates α-syn aggregates, while its downregulation enables their reduction and redeems the PD-like phenotype. Strategies targeting Syn III could thus constitute a therapeutic option for PD.

Can the lack of fibrillar form of alpha-synuclein in Lewy bodies be explained by its catalytic activity?

Finding the causative pathophysiological mechanisms for Parkinson’s disease (PD) is important for developing therapeutic interventions. Until recently, it was believed that Lewy bodies (LBs), the hallmark of PD, are mostly composed of alpha-synuclein (α-syn) fibrils. Recent results (Shahmoradian et al. (2019)) demonstrated that the fibrillar form of α-syn is lacking from LBs. Here we propose that this surprising observation can be explained by the catalytic activity of the fibrillar form of α-syn. We assumed that α-syn fibrils catalyze the formation of LBs, but do not become part of them. We developed a mathematical model based on this hypothesis. By using the developed model, we investigated the consequences of this hypothesis. In particular, the model suggests that the long incubation time of PD can be explained by a two-step aggregation process that leads to its development: (i) aggregation of monomeric α-syn into α-syn oligomers and fibrils and (ii) clustering of membrane-bound organelles, which may cause disruption of axonal trafficking and lead to neuron starvation and death. The model shows that decreasing the rate of destruction of α-syn aggregates in somatic lysosomes accelerates the formation of LBs. Another consequence of the model is the prediction that removing α-syn aggregates from the brain after the aggregation of membrane-bound organelles into LBs has started may not stop the progression of PD because LB formation is an autocatalytic process; hence, the formation of LBs will be catalyzed by aggregates of membrane-bound organelles even in the absence of α-syn aggregates. The performed sensitivity study made it possible to establish the hierarchy of model parameters with respect to their effect on the formation of vesicle aggregates in the soma.

PET tracer for synucleinopathies and neurodegenerative comorbidities with excellent binding affinity and selectivity to α‐synuclein aggregates

AbstractBackgroundThe fundamental role that α‐synuclein (aSyn) plays in the pathogenesis of neurodegenerative synucleinopathies is a well‐accepted fact. Beyond the synucleinopathies, the abnormal deposition of aSyn is frequently seen in a variety of other neurodegenerative proteinopathies including Alzheimer’s disease. We aimed to optimize the pharmacokinetics and the binding properties of disarylbisthiazoles (DABTAs) to aSyn fibrils and versus β‐amyloid (Aβ) and tau (τ) fibrils as PET tracer for synucleinopathies and neurodegenerative comorbidities.MethodWe have identified DABTAs as specific tracer for aSyn in this paper we improved brain uptake kinetics of them by the addition of nitrogen containing heteroaromatic rings in the scaffold. The binding affinity of these ligands were calculated prior using a combination of molecular dynamics and quantum/molecular mechanics. Furthermore, the physicochemical and pharmacokinetics of these DABTAs were modeled using machine learning methods. The radiolabeling via n.c.a one‐ or two‐step F‐18‐fluorination was established. The DABTAs were further evaluated via experimental logD and binding assays, plasma stability, brain metabolite analyses, biodistribution, and for some dynamic PET‐MRI scans.ResultIn silico assays allowed to characterize and evaluate the binding affinity of the candidates, with some of the ligands showing promising affinity and selectivity over Aβ and τ fibrils (80‐120‐fold) based on these calculations. Experimentally, initial binding assays revealed that F‐18‐DABTA‐3 (Log D 2.68), F‐18‐DABTA‐4 (Log D 2.34), F18‐DABTA‐7 (LogD 2.88) and F18‐DABTA‐8 (LogD 2.65) showed good binding affinity to α‐syn fibrils, with some even in the sub‐nanomolar range. Further experiments are ongoing to verify these values. Biodistribution assays in healthy mice brain uptake at 5 mins p.i. ranged from 5.6 ‐ 7.3 %ID/g, with fast brain clearance at 60 mins and 120 mins (up to 0.96 %ID/g & 0.39 %ID/g respectively), with no significant defluorination. The metabolite analyses revealed almost no brain radiometabolites in 20 min.ConclusionIn silico study facilitated the development via a rational design of radioligands with desired characteristics. DABTA results show a lot of promises and encourage us to further investigate them by autoradiography, immunohistochemistry and in vivo imaging in NHP and human.

1-Indanone and 1,3-indandione Derivatives as Ligands for Misfolded α-Synuclein Aggregates.

The development of imaging agents for in vivo detection of alpha-synuclein (α-syn) pathologies faces several challenges. A major gap in the field is the lack of diverse molecular scaffolds with high affinity and selectivity to α-syn fibrils for in vitro screening assays. Better in vitro scaffolds can instruct the discovery of better in vivo agents. We report the rational design, synthesis, and in vitro evaluation of a series of novel 1-indanone and 1,3-indandione derivatives from a Structure-Activity Relationship (SAR) study centered on some existing α-syn fibril binding ligands. Our results from fibril saturation binding experiments show that two of the lead candidates compounds 8 and 32 bind α-syn fibrils with binding constants (Kd ) of 9.0 and 18.8 nM, respectively, and selectivity of greater than 10× for α-syn fibrils compared with amyloid-β (Aβ) and tau fibrils. Our results demonstrate that the lead ligands avidly label all forms of α-syn on PD brain tissue sections, but only the dense core of senile plaques in AD brain tissue, respectively. These results are corroborated by ligand-antibody colocalization data from Syn211, which shows immunoreactivity toward all forms of α-syn aggregates, and Syn303, which displays preferential reactivity toward mature Lewy pathology. Our results reveal that 1-indanone derivatives have desirable properties for the biological evaluation of α-synucleinopathies.

FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.

The hereditary mutation G51D unlocks a distinct fibril strain transmissible to wild-type \u03b1-synuclein

α-Synuclein (α-Syn) can form different fibril strains with distinct polymorphs and neuropathologies, which is associated with the clinicopathological variability in synucleinopathies. How different α-syn fibril strains are produced and selected under disease conditions remains poorly understood. In this study, we show that the hereditary mutation G51D induces α-syn to form a distinct fibril strain in vitro. The cryogenic electron microscopy (cryo-EM) structure of the G51D fibril strain was determined at 2.96 Å resolution. The G51D fibril displays a relatively small and extended serpentine fold distinct from other α-syn fibril structures. Moreover, we show by cryo-EM that wild-type (WT) α-syn can assembly into the G51D fibril strain via cross-seeding with G51D fibrils. Our study reveals a distinct structure of G51D fibril strain triggered by G51D mutation but feasibly adopted by both WT and G51D α-syn, which suggests the cross-seeding and strain selection of WT and mutant α-syn in familial Parkinson’s disease (fPD).