Α-synuclein Fibrils Research Articles

Effect of flavonoids on the destabilization of α-synuclein fibrils and their conversion to amorphous aggregate: A molecular dynamics simulation and experimental study

The second most prevalent neurodegenerative disease, Parkinson's disease (PD), is caused by the accumulation and deposition of fibrillar aggregates of the α-Syn into the Lewy bodies. To create a potent pharmacological candidate to destabilize the preformed α-Syn fibril, it is important to understand the precise molecular mechanism underlying the destabilization of the α-Syn fibril. Through molecular dynamics simulations and experiments, we have examined the molecular mechanisms causing the destabilization and suppression of a newly discovered α-Syn fibril with a Greek-key-like shape and an aggregation prone state (APS) of α-Syn in the presence and absence of various Flvs. According to MD simulation and experimental evidence, morin, quercetin, and myricetin are the Flvs, most capable of destabilizing the fibrils and converting them into amorphous aggregates. Compared to galangin and kaempferol, they have more hydroxyl groups and form more hydrogen bonds with fibrils.The processes by which morin and myricetin prevent new fibril production from APS and destabilize the fibrils are different. According to linear interaction energy analysis, van der Waals interaction predominates with morin, and electrostatic interaction dominates with myricetin. Our MD simulation and experimental findings provide mechanistic insights into how Flvs destabilize α-Syn fibrils and change their morphology, opening the door to developing structure-based drugs for treating Parkinson's disease.

Developmental exposure to the Parkinson's disease-associated organochlorine pesticide dieldrin alters dopamine neurotransmission in α-synuclein pre-formed fibril (PFF)-injected mice.

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.

Elucidation of Secondary Structure and Toxicity of α-Synuclein Oligomers and Fibrils Grown in the Presence of Phosphatidylcholine and Phosphatidylserine.

Abrupt aggregation of α-synuclein (α-Syn) in the midbrain hypothalamus and thalamus is a hallmark of Parkinson's disease (PD), the fastest growing neurodegenerative pathology, projected to strike 12 million people by 2040 worldwide. In this study, we examine the effect of two phospholipids that are present in neuronal membranes, phosphatidylcholine (PC) and phosphatidylserine (PS), on the rate of α-Syn aggregation. We found that PS accelerated α-Syn aggregation, whereas PC strongly inhibited α-Syn aggregation. We also utilized the nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to investigate whether PC and PS only change the rates or also modify the secondary structure of α-Syn aggregates. We found that both phospholipids uniquely altered the secondary structure of α-Syn aggregates present at the lag and growth phase, as well as the late stage of protein aggregation. In addition, compared to the α-Syn aggregates formed in the lipid-free environment, α-Syn:PC and α-Syn:PS aggregates demonstrated higher cellular toxicity to N27 rat neurons. Interestingly, both α-Syn:PC and α-Syn:PS aggregates showed similar levels of oxidative stress, but α-Syn:PC aggregates exhibited a greater degree of mitochondrial dysfunction compared to α-Syn:PS aggregates.

A Fluorescent Probe as a Lead Compound for a Selective α-Synuclein PET Tracer: Development of a Library of 2-Styrylbenzothiazoles and Biological Evaluation of [18F]PFSB and [18F]MFSB.

A method to detect and quantify aggregated α-synuclein (αSYN) fibrils in vivo would drastically impact the current understanding of multiple neurodegenerative diseases, revolutionizing their diagnosis and treatment. Several efforts have produced promising scaffolds, but a notable challenge has hampered the establishment of a clinically successful αSYN positron emission tomography (PET) tracer: the requirement of high selectivity over the other misfolded proteins amyloid β (Aβ) and tau. By designing and screening a library of 2-styrylbenzothiazoles based on the selective fluorescent probe RB1, this study aimed at developing a selective αSYN PET tracer. [3H]PiB competition binding assays identified PFSB (Ki = 25.4 ± 2.3 nM) and its less lipophilic analogue MFSB, which exhibited enhanced affinity to αSYN (Ki = 10.3 ± 4.7 nM) and preserved selectivity over Aβ. The two lead compounds were labeled with fluorine-18 and evaluated using in vitro autoradiography on human brain slices, where they demonstrated up to 4-fold increased specific binding in MSA cases compared to the corresponding control, reasonably reflecting selective binding to αSYN pathology. In vivo PET imaging showed [18F]MFSB successfully crosses the blood-brain barrier (BBB) and is taken up in the brain (SUV = 1.79 ± 0.02). Although its pharmacokinetic profile raises the need for additional structural optimization, [18F]MFSB represents a critical step forward in the development of a successful αSYN PET tracer by overcoming the major challenge of αSYN/Aβ selectivity.

Coumarin-chalcone hybrid LM-021 and indole derivative NC009-1 targeting inflammation and oxidative stress to protect BE(2)-M17 cells against α-synuclein toxicity.

Parkinson's disease (PD) is featured mainly by the loss of dopaminergic neurons and the presence of α-synuclein-containing aggregates in the substantia nigra of brain. The α-synuclein fibrils and aggregates lead to increased oxidative stress and neural toxicity in PD. Chronic inflammation mediated by microglia is one of the hallmarks of PD pathophysiology. In this report, we showed that coumarin-chalcone hybrid LM-021 and indole derivative NC009-1 reduced the expression of major histocompatibility complex-II, NLR family pyrin domain containing (NLRP) 3, caspase-1, inducible nitric oxide synthase, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in α-synuclein-activated mouse BV-2 microglia. Release of pro-inflammatory mediators including nitric oxide, IL-1β, IL-6 and TNF-α was also mitigated. In BE(2)-M17 cells expressing A53T α-synuclein aggregates, LM-021 and NC009-1 reduced α-synuclein aggregation, neuroinflammation, oxidative stress and apoptosis, and promoted neurite outgrowth. These protective effects were mediated by downregulating NLRP1, IL-1β and IL-6, and their downstream pathways including nuclear factor (NF)-κB inhibitor alpha (IκBα)/NF-κB P65 subunit (P65), c-Jun N-terminal kinase (JNK)/proto-oncogene c-Jun (JUN), mitogen-activated protein kinase 14 (P38)/signal transducer and activator of transcription (STAT) 1, and Janus kinase 2 (JAK2)/STAT3. The study results indicate LM-021 and NC009-1 as potential new drug candidates for PD.

The Poly-Arginine Peptide R18D Interferes with the Internalisation of α-Synuclein Pre-Formed Fibrils in STC-1 Enteroendocrine Cells.

In Parkinson's disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 μM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology.

Identification of Clec7a as the therapeutic target of rTMS in alleviating Parkinson's disease: targeting neuroinflammation

Parkinson's disease (PD) is a neurodegenerative disease. Repetitive transcranial magnetic stimulation (rTMS) is a therapeutic tool in PD. High-throughput sequencing was performed to screen potential therapeutic targets in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. The candidate gene, Clec7a, was screened out and validated. Clec7a is a pattern recognition receptor involved in neuroinflammation. The higher expression of Clec7a was observed in the substantia nigra (SN) and striatum of PD rats with dopaminergic neurons damage and was mainly localized in the microglial. Adeno-associated virus (AAV)-mediated specific knockdown of Clec7a in microglial alleviated 6-OHDA induced motor deficits and nigrostriatal dopaminergic neuron damage of rats, as evidenced by the increase of tyrosine hydroxylase (TH) -positive neurons in SN, as well as dopaminergic nerve fibers in the striatum. Clec7a knockdown restrained the neuroinflammation by suppressing inflammatory factors (IFN-γ, TNF-α, IL-1β, IL-18, and IL-6) release in SN, which might result from enhanced Arg-1 expression (M2 polarization) and defective inducible nitric oxide synthase (iNOS) expression (M1 polarization). The same phenomena were also observed in the LPS inflammatory rat model of PD. In vitro, α-synuclein fibrils induced upregulation of Clec7a expression and microglia polarization to a pro-inflammatory state of BV2 cells, leading to increased release of cytokines. However, Clec7a knockdown reversed those changes and induced a shift to an anti-inflammatory phenotype in BV2 cells. In conclusion, our study suggested that Clec7a was involved in PD pathogenesis, and its inhibition might protect rats from PD by depressing neuroinflammation through microglial polarization.

Probing the molecular mechanisms of α-synuclein inhibitors unveils promising natural candidates through machine-learning QSAR, pharmacophore modeling, and molecular dynamics simulations.

Parkinson's disease is characterized by a multifactorial nature that is linked to different pathways. Among them, the abnormal deposition and accumulation of α-synuclein fibrils is considered a neuropathological hallmark of Parkinson's disease. Several synthetic and natural compounds have been tested for their potency to inhibit the aggregation of α-synuclein. However, the molecular mechanisms responsible for the potency of these drugs to further rationalize their development and optimization are yet to be determined. To enhance our understanding of the structural requirements necessary for modulating the aggregation of α-synuclein fibrils, we retrieved a large dataset of α-synuclein inhibitors with their reported potency from the ChEMBL database to explore their chemical space and to generate QSAR models for predicting new bioactive compounds. The best performing QSAR model was applied to the LOTUS natural products database to screen for potential α-synuclein inhibitors followed by a pharmacophore design using the representative compounds sampled from each cluster in the ChEMBL dataset. Five natural products were retained after molecular docking studies displaying a binding affinity of - 6.0kcal/mol or lower. ADMET analysis revealed satisfactory properties and predicted that all the compounds can cross the blood-brain barrier and reach their target. Finally, molecular dynamics simulations demonstrated the superior stability of LTS0078917 compared to the clinical candidate, Anle138b. We found that LTS0078917 shows promise in stabilizing the α-synuclein monomer by specifically binding to its hairpin-like coil within the N-terminal region. Our dynamic analysis of the inhibitor-monomer complex revealed a tendency towards a more compact conformation, potentially reducing the likelihood of adopting an elongated structure that favors the formation and aggregation of pathological oligomers. These findings offer valuable insights for the development of novel α-synuclein inhibitors derived from natural sources.

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

Structural mechanism for specific binding of chemical compounds to amyloid fibrils.

Amyloid fibril is an important pharmaceutical target for diagnostic and therapeutic treatment of neurodegenerative diseases. However, rational design of chemical compounds that interact with amyloid fibrils is unachievable due to the lack of mechanistic understanding of the ligand-fibril interaction. Here we used cryoelectron microscopy to survey the amyloid fibril-binding mechanism of a series of compounds including classic dyes, (pre)clinical imaging tracers and newly identified binders from high-throughput screening. We obtained clear densities of several compounds in complex with an α-synuclein fibril. These structures unveil the basic mechanism of the ligand-fibril interaction, which exhibits remarkable difference from the canonical ligand-protein interaction. In addition, we discovered a druggable pocket that is also conserved in the ex vivo α-synuclein fibrils from multiple system atrophy. Collectively, these findings expand our knowledge of protein-ligand interaction in the amyloid fibril state, which will enable rational design of amyloid binders in a medicinally beneficial way.

Monitoring α-synuclein Aggregation Induced by Preformed α-synuclein Fibrils in an In Vitro Model System.

Parkinson's disease (PD) is characterized by the presence of α-synuclein (α-syn) inclusions in the brain and the degeneration of dopamine-producing neurons. There is evidence to suggest that the progression of PD may be due to the prion-like spread of α-syn aggregates, so understanding and limiting α-syn propagation is a key area of research for developing PD treatments. Several cellular and animal model systems have been established to monitor α-syn aggregation and propagation. In this study, we developed an in vitro model using A53T α-syn-EGFP overexpressing SH-SY5Y cells and validated its usefulness for high-throughput screening of potential therapeutic targets. Treatment with preformed recombinant α-syn fibrils induced the formation of aggregation puncta of A53T α-syn-EGFP in these cells, which were analyzed using four indices: number of dots per cell, size of dots, intensity of dots, and percentage of cells containing aggregation puncta. Four indices are reliable indicators of the effectiveness of interventions against α-syn propagation in a one-day treatment model to minimize the screening time. This simple and efficient in vitro model system can be used for high-throughput screening to discover new targets for inhibiting α-syn propagation.

Cerebral Microvascular Injury Induced by Lag3-Dependent α-Synuclein Fibril Endocytosis Exacerbates Cognitive Impairment in a Mouse Model of α-Synucleinopathies.

The pathological accumulation of α-synuclein (α-Syn) and the transmission of misfolded α-Syn underlie α-synucleinopathies. Increased plasma α-Syn levels are associated with cognitive impairment in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies, but it is still unknown whether the cognitive deficits in α-synucleinopathies have a common vascular pathological origin. Here, it is reported that combined injection of α-Syn preformed fibrils (PFFs) in the unilateral substantia nigra pars compacta, hippocampus, and cerebral cortex results in impaired spatial learning and memory abilities at 6 months post-injection and that this cognitive decline is related to cerebral microvascular injury. Moreover, insoluble α-Syn inclusions are found to form in primary mouse brain microvascular endothelial cells (BMVECs) through lymphocyte-activation gene 3 (Lag3)-dependent α-Syn PFFs endocytosis, causing poly(ADP-ribose)-driven cell death and reducing the expression of tight junction proteins in BMVECs. Knockout of Lag3 in vitro prevents α-Syn PFFs from entering BMVECs, thereby reducing the abovementioned response induced by α-Syn PFFs. Deletion of endothelial cell-specific Lag3 in vivo reverses the negative effects of α-Syn PFFs on cerebral microvessels and cognitive function. In short, this study reveals the effectiveness of targeting Lag3 to block the spread of α-Syn fibrils to endothelial cells in order to improve cognition.

A 14-day pulse of PLX5622 modifies α-synucleinopathy in preformed fibril-infused aged mice of both sexes

Reactive microglia are observed with aging and in Lewy body disorders, including within the olfactory bulb of men with Parkinson's disease. However, the functional impact of microglia in these disorders is still debated. Resetting these reactive cells by a brief dietary pulse of the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 may hold therapeutic potential against Lewy-related pathologies. To our knowledge, withdrawal of PLX5622 after short-term exposure has not been tested in the preformed α-synuclein fibril (PFF) model, including in aged mice of both sexes. Compared to aged female mice, we report that aged males on the control diet showed higher numbers of phosphorylated α-synuclein+ inclusions in the limbic rhinencephalon after PFFs were injected in the posterior olfactory bulb. However, aged females displayed larger inclusion sizes compared to males. Short-term (14-day) dietary exposure to PLX5622 followed by control chow reduced inclusion numbers and levels of insoluble α-synuclein in aged males—but not females—and unexpectedly raised inclusion sizes in both sexes. Transient delivery of PLX5622 also improved spatial reference memory in PFF-infused aged mice, as evidenced by an increase in novel arm entries in a Y-maze. Superior memory was positively correlated with inclusion sizes but negatively correlated with inclusion numbers. Although we caution that PLX5622 delivery must be tested further in models of α-synucleinopathy, our data suggest that larger-sized—but fewer—α-synucleinopathic structures are associated with better neurological outcomes in PFF-infused aged mice.

Cellular processing of α-synuclein fibrils results in distinct physiological C-terminal truncations with a major cleavage site at residue Glu 114

α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both invitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. Invitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.

Sortilin acts as an endocytic receptor for α-synuclein fibril.

Cell-to-cell spreading of misfolded α-synuclein (αSYN) is supposed to play a key role in the pathological progression of Parkinson's disease (PD) and other synucleinopathies. Receptor-mediated endocytosis has been shown to contributes to the uptake of αSYN in both neuronal and glial cells. To determine the receptor involved in αSYN endocytosis on the cell surface, we performed unbiased, and comprehensive screening using a membrane protein library of the mouse whole brain combined with affinity chromatography and mass spectrometry. The candidate molecules hit in the initial screening were validated by co-immunoprecipitation using cultured cells; sortilin, a vacuolar protein sorting 10 protein family sorting receptor, exhibited the strongest binding to αSYN fibrils. Notably, the intracellular uptake of fibrillar αSYN was slightly but significantly altered, depending on the expression level of sortilin on the cell surface, and time-lapse image analyses revealed the concomitant internalization and endosomal sorting of αSYN fibrils and sortilin. Domain deletion in the extracellular portion of sortilin revealed that the ten conserved cysteines (10CC) segment of sortilin was involved in the binding and endocytosis of fibrillar αSYN; importantly, pretreatment with a 10CC domain-specific antibody significantly hindered αSYN fibril uptake. The presence of sortilin in the core structure of Lewy bodies and glial cytoplasmic inclusions in the brain of synucleinopathy patients was confirmed via immunohistochemistry, and the expression level of sortilin in mesencephalic dopaminergic neurons may be altered with disease progression. These results provide compelling evidence that sortilin acts as an endocytic receptor for pathogenic form of αSYN, and yields important insight for the development of disease-modifying targets for synucleinopathies.

Retina-to-brain spreading of α-synuclein after intravitreal injection of preformed fibrils

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the aggregation of misfolded α-synuclein and progressive spreading of the aggregates from a few discrete regions to wider brain regions. Although PD has been classically considered a movement disorder, a large body of clinical evidence has revealed the progressive occurrence of non-motor symptoms. Patients present visual symptoms in the initial stages of the disease, and accumulation of phospho-α-synuclein, dopaminergic neuronal loss, and retinal thinning has been observed in the retinas of PD patients. Based on such human data, we hypothesized that α-synuclein aggregation can initiate in the retina and spread to the brain through the visual pathway. Here, we demonstrate accumulation of α-synuclein in the retinas and brains of naive mice after intravitreal injection of α-synuclein preformed fibrils (PFFs). Histological analyses showed deposition of phospho-α-synuclein inclusions within the retina 2 months after injection, with increased oxidative stress leading to loss of retinal ganglion cells and dopaminergic dysfunction. In addition, we found accumulation of phospho-α-synuclein in cortical areas with accompanying neuroinflammation after 5 months. Collectively, our findings suggest that retinal synucleinopathy lesions initiated by intravitreal injection of α-synuclein PFFs spread to various brain regions through the visual pathway in mice.

Sesamol as a potential small molecule in inhibiting the induction of neurotoxic amyloids fibrils involved in the pathophysiology of alzheimer's disease

The presence of possibly toxic forms of intracellular α-synuclein, binding of aggregated α-synuclein and monomeric Aβ, and the potential co-localization of α-synuclein with intracellular neurofibrillary tangles as well as Aβ plaques in the brain could be the main causes in serious clinical symptoms mostly developed in Alzheimer’s disease (AD) patients. Amyloid formation of α-synuclein can also induce neurotoxic effects through stimulation of endoplasmic reticulum stress (ERS). Therefore, the inhibition of protein aggregation can be of great importance for the control of pathophysiology of AD. Here, we assessed the use of sesamol as bioactive small molecule in inhibition of α-synuclein fibrillogenesis and neurotoxicity by different biophysical [ThT/Nile red/Congo red (CR)/circular dichroism (CD)] and cellular (MTT and qRT-PCR) analyses. From the spectroscopic analyses, we found that sesamol displays potential effect on the inhibiting the α-synuclein fibril formation. Also, sesamol showed no significant neurotoxicity and co-incubation of α-synuclein with sesamol apparently mitigated the ERS-mediated apoptosis induced by α-synuclein amyloids through regulation of IRE1, PERK, ATF6, and caspase-3 mRNA. Overall, sesamol-based compounds can be further developed and assessed for the regulation of pathophysiology of AD in which α-synuclein amyloids are predominantly involved.

α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson’s disease patient’s brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.

The Structural Combination of SIL and MODAG Scaffolds Fails to Enhance Binding to α-Synuclein but Reveals Promising Affinity to Amyloid β.

A technique to image α-synuclein (αSYN) fibrils in vivo is an unmet scientific and clinical need that would represent a transformative tool in the understanding, diagnosis, and treatment of various neurodegenerative diseases. Several classes of compounds have shown promising results as potential PET tracers, but no candidate has yet exhibited the affinity and selectivity required to reach clinical application. We hypothesized that the application of the rational drug design technique of molecular hybridization to two promising lead scaffolds could enhance the binding to αSYN up to the fulfillment of those requirements. By combining the structures of SIL and MODAG tracers, we developed a library of diarylpyrazoles (DAPs). In vitro evaluation through competition assays against [3H]SIL26 and [3H]MODAG-001 showed the novel hybrid scaffold to have preferential binding affinity for amyloid β (Aβ) over αSYN fibrils. A ring-opening modification on the phenothiazine building block to produce analogs with increased three-dimensional flexibility did not result in an improved αSYN binding but a complete loss of competition, as well as a significant reduction in Aβ affinity. The combination of the phenothiazine and the 3,5-diphenylpyrazole scaffolds into DAP hybrids did not generate an enhanced αSYN PET tracer lead compound. Instead, these efforts identified a scaffold for promising Aβ ligands that may be relevant to the treatment and monitoring of Alzheimer's disease (AD).

Inhibition of primary and secondary nucleation alongwith disruption of amyloid fibrils and alleviation of associated cytotoxicity: A biophysical insight of a novel property of Chlorpropamide (an anti-diabetic drug)

Aggregation of physiologically synthesized soluble proteins to insoluble, cytotoxic fibrils is a pre-requisite for pathogenesis of amyloid associated disorders including Alzheimer's disease, non-systemic amyloidosis, Parkinson's disease, etc. Considerable advancement has been made to understand the mechanism behind aggregation process but till date we have no efficient cure and preventive therapy for associated diseases. Strategies to prevent protein aggregation are nevertheless many which have been proved promisingly successful in vitro. One of those is repurposing already approved drugs that saves time and money too and has been employed in this study. Here, for the first time we are reporting the effectiveness of an anti-diabetic drug chlorpropamide (CHL) under dosage conditions, a novel property to inhibit aggregation in human lysozyme (HL) in vitro. Spectroscopic (Turbidity, RLS, ThT, DLS, ANS) and microscopic (CLSM) results demonstrates that CHL has the potency to suppress aggregation in HL up to 70 %. CHL is shown to affect the elongation of fibrils with IC50 value of 88.5 μM as clear from the kinetics results, may be by interacting near/with aggregation prone regions of HL. Hemolytic assay also revealed the reduced cytotoxicity in the presence of CHL. Disruption of amyloid fibrils and inhibition of secondary nucleation in the presence of CHL was also evidenced by ThT, CD and CLSM results with reduced cytotoxicity as confirmed by hemolytic assay. We also performed preliminary studies on α-synuclein fibrillation inhibition and surprisingly found that CHL is not just inhibiting the fibrillation but also stabilizing the protein in its native state. These findings insinuate that CHL (anti-diabetic) possess multiple roles and can be a promising drug for developing therapeutic against non-systemic amyloidosis, Parkinson's disease and other amyloid associated disorders.