Preformed Fibrils Research Articles

ASC specks exacerbate α‑synuclein pathology via amplifying NLRP3 inflammasome activities

BackgroundInflammasome activation has a pathogenic role in Parkinson’s disease (PD). Up-regulated expressions of inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and assembly of ASC specks have been observed in postmortems of human PD brains and experimental PD models. Extracellular ASC specks behave like danger signals and sustain prolonged inflammasome activation. However, the contribution of ASC specks in propagation of inflammasome activation and pathological progression in PD has not been fully established.MethodsHerein, we used human A53T mutant α-synuclein preformed fibrils (PFFs)-stimulated microglia in vitro and unilateral striatal stereotaxic injection of PFFs-induced mice model of PD in vivo, to investigate the significance of ASC specks in PD pathological progression. Rotarod and open-field tests were performed to measure motor behaviors of indicated mice. Changes in the molecular expression were evaluated by immunofluorescence and immunoblotting (IB). Intracellular knockdown of the ASC in BV2 cells was performed using si-RNA. Microglial and neuronal cells were co-cultured in a trans-well system to determine the effects of ASC knockdown on cytoprotection.ResultsWe observed a direct relationship between levels of ASC protein and misfolded α‑synuclein aggregates in PD mice brains. ASC specks amplified NLRP3 inflammasome activation driven by α-synuclein PFFs stimulation, which aggravated reactive microgliosis and accelerated α‑synuclein pathology, dopaminergic neurodegeneration and motor deficits. Endogenous ASC knockdown suppressed microglial inflammasome activation and neuronal α‑synuclein aggregation.ConclusionsIn conclusion, our study elucidated that ASC specks contribute to the propagation of inflammasome activation-associated α‑synuclein pathology in PD, which forms the basis for targeting ASC as a potential therapy for PD.

Differential binding dynamics of four microtubule-binding repeats of tau with an amyloid-β fibril seed.

Both senile plaques formed by amyloid-beta (Aβ) and neurofibrillary tangles comprised of tau are hallmarks of Alzheimer's disease (AD). The accumulation of neurofibrillary tangles better correlates with the loss of cognitive function than senile plaques, but neurofibrillary tangles are rarely observed without the presence of senile plaques. Hence, cross-seeding of tau by preformed Aβ amyloid fibril seeds has been proposed to drive the aggregation of tau and exacerbate AD progression, but the molecular mechanism remains poorly understood. Herein, we identified cross-interaction hotspots between Aβ and tau using atomistic discrete molecular dynamics simulations (DMD) and confirmed the critical role of the four microtubule-binding repeats of tau (R1-R4) in the cross-interaction with Aβ. We further investigated the binding structure and dynamics of each tau repeat with a preformed Aβ fibril seed. Specifically, R1 and R3 preferred to bind the lateral instead of elongation surfaces of the fibril, preserving similar secondary structures as in the solution. In contrast, R2 and R4 had higher binding propensities to the elongation than lateral surfaces, adopting β-sheet structures by forming hydrogen bonds with the exposed hydrogen bond donors and acceptors. Together, our results suggest that four MTBD repeats play distinct roles in driving the binding of tau to two different surfaces of an Aβ fibril seed. The binding of tau to the lateral surface of Aβ fibril can increase the local concentration, while the binding in the elongation surface promotes β-sheet formation, both of which reduce the free energy barrier for tau aggregation nucleation and subsequent fibrillization. Our computational study offers molecular insight into the experimentally observed cross-seeding of Tau aggregation by pre-formed Aβ fibril seeds and helps better understand the pathological synergy of Aβ and tau aggregation in the etiology of AD.

Cyclic-NDGA effectively inhibits human gamma-synuclein fibrillation, forms off-pathway species, and disintegrates pre-formed mature fibrils.

Parkinson's disease is one of the various diseases resulting from protein misfolding, aggregation and fibrillation and is characterized by Lewy body deposits which contain the protein α-synuclein (α-syn) as their major component. γ-synuclein (γ-syn), one of the three synucleins is also present in the Lewy bodies along with α-syn and is also implicated in various types of cancers especially breast cancer. It is known to seed the fibrillation of α-syn after its oxidation at methionine residue leading to synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule therapeutics against γ-syn fibrillation remains largely unexplored. We have investigated the modulatory properties of cyclic-Nordihydroguaiaretic acid (cNDGA), a plant polyphenol, on the structural and aggregational properties of human γ-syn employing various techniques namely ThT fluorescence, Rayleigh Light scattering, ANS binding, far-UV CD and FTIR spectroscopy, atomic force microscopy and MTT assay. cNDGA inhibits the fibrillation of γ-syn and unlike mature control fibrils, γ-syn forms large aggregates with no ThT binding property, negligible hydrophobicity, with lesser β-sheet content and reduced cytotoxicity when incubated with cNDGA. These species formed as a result of weak interaction of γ-syn with cNDGA (Kd ∼10−5M) as investigated by isothermal titration calorimetry are off pathway in nature suggested by seeding experiments. Increasing concentration of cNDGA resulted in an increased recovery of monomeric γ-syn as shown by SDS-PAGE and Native-PAGE. cNDGA was most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and was also capable of disintegrating the pre-formed mature fibrils. It can be concluded that cNDGA effectively suppresses γ-syn fibrillation and forms species that are less toxic in nature. These findings could be further explored for the use of cNDGA and similar flavonoids as therapeutics against γ-synucleinopathies.

Direct observation of single-molecular hIAPP fibril elongation reveal molecular mechanisms of morphological dependence and asymmetric growth.

Rapid growth of amyloid fibrils via seeded conformational conversion of monomers, is a critical step of fibrillization and important for the prion-like transmission of amyloid diseases. Amyloid fibrils are often polymorphic with relative populations morphology-dependent, but the molecular mechanisms of fibril elongation and corresponding morphological dependence remain poorly understood. Here, we applied replica exchange all-atom discrete molecular dynamics simulations to study the conformational conversion of hIAPP, an amyloid peptide associated with type-2 diabetes, catalyzed by two representative fibrils with distinct morphologies where hIAPP adopted S-shaped and extended conformations correspondingly. For both cases, the incorporation of monomers into the preformed fibrils with parallel in-register alignments was observed in our unbiased simulations. The fibril elongation free energy landscape derived from simulations is characterized by multiple basins corresponding to different intermediate states. Fibril morphology affected monomer binding at fibril elongation and lateral surfaces, and also the conformational conversion dynamics at the elongation surface, resulting into different elongation rates and thus relative populations for different fibril morphologies as observed experimentally. We also investigated the dynamics of hIAPP monomers bound to the fibril elongation surfaces at opposite ends. We observed distinct free energy landscapes in agreement with experimentally-observed asymmetric fibril growth, which can be attributed to subtle physicochemical difference of two ends in terms of contacting surfaces and patterns of available hydrogen bond donors and acceptors. Together, our results offered molecular insights to seeded conformational conformation of fibril elongation, morphological dependence, asymmetry fibril growth, and could shed light on amyloid strains with distinct disease progression.

The purely thermodynamic anti-prionic mode of action for the treatment of neurodegenerative diseases - realized by target protein specific all-D-peptides.

Neurodegenerative protein-misfolding diseases, like Alzheimer's (AD) and Parkinson's disease (PD), are driven by prion-like self-replicating and propagating protein assemblies of Aβ, α-synuclein, and many more. Therefore, we have developed the so-called anti-prionic mode of action (MoA). It is realized by all-D-enantiomeric peptide ligands that bind the monomeric protein of interest with high affinity, thereby stabilizing the physiological intrinsically disordered monomer structure. We call this purely thermodynamic driven mode of action “anti-prionic”, because it is eliminating already existing prion-like aggregates by disassembling them into non-toxic and functional monomers. I will summarize the current, and yet unpublished, progress in realizing the anti-prionic MoA for the target proteins α-synuclein and Aβ. Briefly, the most promising all-D-enantiomeric ligand for α-synuclein, SVD-1a, disassembles preformed α-synuclein fibrils (PFF) as shown by AFM, DLS and SEC analysis. SPR and NMR demonstrate picomolar affinity of SVD-1a to α-synuclein monomers, while keeping them in their physiological intrinsically disordered conformation. The all-D-enantiomeric ligand for Aβ, RD2, disassembles Aβ oligomers obtained from brain tissue of former AD patients. A clinical phase Ib, double-bind, placebo-controlled study with 20 ATN positive patients treated once daily orally with RD2 or placebo for 4 weeks with an additional 4 weeks follow up period demonstrates good safety and tolerability, and yields interesting results on efficacy. This is valuable information for the scheduled phase II study. I will also acknowledge the many contributors of both developments that are too many to be included here in the abstract. Conclusion: The unique anti-prionic mode of action for the treatment of AD, PD and other protein misfolding diseases is promising.

Studies on alpha-synuclein and islet amyloid polypeptide interaction.

Introduction: Parkinson's disease and type 2 diabetes have both elements of local amyloid depositions in their pathogenesis. In Parkinson's disease, alpha-synuclein (aSyn) forms insoluble Lewy bodies and Lewy neurites in brain neurons, and in type 2 diabetes, islet amyloid polypeptide (IAPP) comprises the amyloid in the islets of Langerhans. In this study, we assessed the interaction between aSyn and IAPP in human pancreatic tissues, both ex vivo and in vitro. Material and Methods: The antibody-based detection techniques, proximity ligation assay (PLA), and immuno-TEM were used for co-localization studies. Bifluorescence complementation (BiFC) was used for interaction studies between IAPP and aSyn in HEK 293 cells. The Thioflavin T assay was used for studies of cross-seeding between IAPP and aSyn. ASyn was downregulated with siRNA, and insulin secretion was monitored using TIRF microscopy. Results: We demonstrate intracellular co-localization of aSyn with IAPP, while aSyn is absent in the extracellular amyloid deposits. ASyn reactivity is present in the secretory granules of β-cells and some α-cells in human islets. The BiFC-expression of aSyn/aSyn and IAPP/IAPP in HEK293 cells resulted in 29.3% and 19.7% fluorescent cells, respectively, while aSyn/IAPP co-expression resulted in ∼10% fluorescent cells. Preformed aSyn fibrils seeded IAPP fibril formation in vitro, but adding preformed IAPP seeds to aSyn did not change aSyn fibrillation. In addition, mixing monomeric aSyn with monomeric IAPP did not affect IAPP fibril formation. Finally, the knockdown of endogenous aSyn did not affect β cell function or viability, nor did overexpression of aSyn affect β cell viability. Discussion: Despite the proximity of aSyn and IAPP in β-cells and the detected capacity of preformed aSyn fibrils to seed IAPP in vitro, it is still an open question if an interaction between the two molecules is of pathogenic significance for type 2 diabetes.

Tracking the Neurodegeneration and Behavioral Changes in Mice Model of Prodromal Phase Alpha-Synucleinopathy


 
 
 Background:
 The aggregation of misfolded alpha-synuclein (αSyn) has been shown to cause neurodegeneration in a mouse model and lead to behavioral changes. While others have examined end-stage (after neurodegeneration has set in) behavioral changes in mice after αSyn preformed fibrils (PFF) seeding, the precise timeline of circuit changes and associated behavioral deficits during the prodromal phase of synucleinopathic spread is poorly understood. We set out to measure and characterize the extent to which behavior is affected by primary motor cortex (M1) PFF seeding in mice.
 Experimental Design:
 We quantified the neuronal cell body density at M1 layers 2/3 and 5 using immunohistochemistry cell assays to determine if there were neurodegenerative effects under the PFF seeding strategy. Furthermore, using Bonsai, a visual reactive programming language, we tracked the behavior of the PFF seeded mice for exploratory motives, decreases in which can be used to show cognitive decline. To further ascertain the effect of αSyn on vulnerable excitatory pathways in the cortex, we performed prodromal injections of αSyn PFF into the M1 of ChATCre mice along with projection tracing in the basal forebrain.
 Results:
 During the first 3 months following PFF seeding, the density of neuronal cell bodies in M1 layers 2/3 and 5 remains unchanged in PFF seeded mice compared to control mice. The exploratory index of PFF seeded mice was comparable to that of control mice. These results suggest insignificant neurodegeneration and behavioral change in the first 3 months following PFF seeding.
 Conclusion:
 Work done previously in this lab tracking the spread of PFF in M1 shows significant spread of αSyn for 3 months following prodromal injection. The results from this work show that there is little neurodegeneration or behavioral change in that timeframe despite the significant spread of αSyn. Future work hinges on further probing this phenomenon.
 
 

Self-Assembly of Amyloid Fibrils into 3D Gel Clusters versus 2D Sheets.

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this "secondary" self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril-fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly.

Mesalazine Inhibits Amyloid Formation and Destabilizes Pre-formed Amyloid Fibrils in the Human Insulin.

Amyloid formation due to protein aggregation is associated with several amyloid diseases (amyloidosis). The use of small organic ligands as inhibitors of protein aggregation is an attractive strategy for the treatment of these diseases. In the present study, we evaluated the in vitro inhibitory and destabilizing effects of Mesalazine on human insulin fibrillation. To induce fibrillation, human insulin was incubated in 50mM glycine buffer (pH 2.0) at 50°C. The effect of Mesalazine on insulin amyloid aggregation was studied using spectroscopic, imaging, and computational approaches.Based on the results, the Mesalazine in a concentration-dependent manner (different ratios (1:0.1, 1:0.5, 1:1, and 1:5) of the insulin to Mesalazine) prevented the formation of amyloid fibrils and destabilized pre-formed fibrils. In addition, our molecular docking study confirmed the binding of Mesalazine to insulin through hydrogen bonds and hydrophobic interactions. Our findings suggest that Mesalazine may have therapeutic potential in the prevention of insulin amyloidosis and localized amyloidosis.

Pharmacological characterization of the small molecule 03A10 as an inhibitor of α-synuclein aggregation for Parkinson's disease treatment.

Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson's disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 μM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3mg· kg-1 ·d-1, 1 mg ·kg-1 ·d-1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg-1 ·d-1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, inan MPTP/p mouse model, and PFF-inoculated mice.

Rodent Model Preclinical Assessment of PEGylated Block Copolymer Targeting Cognition and Oxidative Stress Insults of Alzheimer's Disease.

Misfolded peptide amyloid beta (Aβ42), neurofibrillary tangles of hyper-phosphorylated tau, oxidative damage to the brain, and neuroinflammation are distinguished determinants of Alzheimer's disease (AD) responsible for disease progression. This multifaceted neurodegenerative disease is challenging to cure under a single treatment regime until the key disease determinants are traced for their sequential occurrence in disease progression. In an early report, a novel side-chain tripeptide containing PEGylated block copolymer has been tested thoroughly in vitro and in silico for the early inhibition of Aβ42 aggregation as well as degradation of preformed Aβ42 fibril deposits. The present study demonstrates a preclinical assessment of the PEGylated block copolymer in colchicine-induced AD-mimicking rodent model. The colchicine-induced Wistar rats receiving an intranasal delivery of the block copolymer at a daily dosage of 100µg/kg and 200µg/kg body weights, respectively, for 14days manifested a notable attenuation of behavioral deficit pattern, oxidative stress, and neurotransmitters' deficiency as compared to the untreated ones. The current study also reports the ameliorative property of the PEGylated compound for progressive neuroinflammation and decreased mitochondrial bioenergetics in astrocytoma cell line, viz., U87. A closer look into the drug mechanism of action of a compact 3D PEGylated block copolymer confirmed its disintegrative interaction with Aβ42 fibril via in silico simulation. The results obtained from this study signify the potential of the novel PEGylated block copolymer to ameliorate the cognitive decline and progressive oxidative insults in AD and may envision a successful clinical phase trial. The amelioration of disease condition of colchicine-induced AD rat. Initially the rat has given colchicine via stereotaxic surgery which led to a mimicking condition of AD including neuronal death in hippocampal CA1 region. After recovery from the surgery, the rat was treated with the PEGylated block copolymer through intranasal delivery, and this has led to the decrease in neuronal death in hippocampal CA1 region. The mechanism of drug action has shown by the separation of monomer chains of Aβ42.

Mechanism of the interaction of toxic SOD1 fibrils with two potent polyphenols: curcumin and quercetin.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease commonly caused due to the aggregation of superoxide dismutase 1 (SOD1) protein. Finding inhibitors of SOD1 aggregation is of prime concern, but understanding the mechanistic action of inhibitors is equally important. Recent experiments found that two polyphenols, curcumin, and quercetin, have the ability to inhibit SOD1 aggregation. Quercetin was experimentally proven to break pre-formed fibrils into shorter segments, while curcumin did not significantly affect the pre-formed species. Here, we delve deeper into understanding the mechanism of action of quercetin and curcumin on pre-formed octameric fibrils of SOD1 (28PVKVWGSIKGL38: chains A-H) with the help of molecular dynamics (MD) simulations of a fibril docked polyphenol complex. Our results suggest that quercetin shows π-π stacking interaction with one of the key residues for toxic amyloid formation, Trp 32 of chains D, E, and F, and breaks the peptide chains G, and H from the rest of the fibril. On the other hand, curcumin binds to the hydrophobic amino acids of almost all the chains B-H and stabilizes the fibril rather than destabilizing it. Binding free energy calculations using MM/PBSA showed that curcumin binds more strongly to the SOD1 fibril due to greater van der Waals interactions compared to quercetin. These findings provide insights for the development of potential ALS treatments.

The formation mechanism of lewy body at DMV related to parkinson’s disease

The purpose of this experiment is to demonstrate the mechanism of Lewy body aggregation and formation in DMV by gene modification and injection of exogenous preformed fibrils, and how this relates to Parkinson's disease. Through a series of work such as cardiac perfusion, frozen section, and immunohistochemical staining, the existence of cholinergic neurons and Lewy bodies at the DMV was finally confirmed by 63x laser confocal microscopy, which explained the symptoms of the digestive system during the early stage of Parkinson's disease.

Insights into the remarkable attenuation of hen egg white lysozyme amyloid fibril formation mediated by biogenic gold nanoparticles stabilized by quercetin-functionalized tara gum

Aberrant protein misfolding and/or aggregation and fibrillation has been linked to the pathogenesis of several debilitating chronic diseases including Alzheimer's and Parkinson's disease. Inhibiting protein amyloidogenesis has been proposed as a viable strategy to prevent or ameliorate associated disorders. Herein, we investigated the anti-amyloidogenic properties of biogenic gold nanoparticles (QTG-GNP) prepared via a simple green chemistry route and stabilized by quercetin-functionalized tara gum (QTG). The synthesized QTG-GNP was extensively characterized for its physicochemical attributes via UV–visible spectroscopy, TEM, FESEM, EDX, DLS/Zeta potential, FTIR, RAMAN, XRD, XPS, and TGA analyses, as well as for its biological properties. The results revealed that small-sized (5.01 ± 1.17 nm), well-dispersed, highly stable and round-shaped biogenic gold nanoparticles were successfully synthesized at room temperature with QTG as the sole reductant /stabilizer. Importantly, QTG-GNP demonstrated potent anti-aggregation and fibrillation inhibitory effects against amyloidogenic hen egg white lysozyme (HEWL). Also, QTG-GNP was able to dissociate pre-formed HEWL amyloid fibrils. Furthermore, the constructed nanoparticles exhibited potent anti-radical activities against DPPH and ABTS+ and were cytocompatible with mouse L929 fibroblast cells. On the basis of these findings, it was established that QTG-GNP holds strong prospects for further development as an agent for countering protein aggregation and associated disease conditions.

Yeast-derived vacuoles as a novel carrier with enhanced hCMEC/D3 cell monolayer penetration.

The blood-brain barrier (BBB) is a brain protection structure that restricts drug delivery from the blood to the central nervous system. Thus, we developed a novel drug carrier using yeast vacuoles to overcome this problem. The purpose of this study was to assess the drug transportability of yeast vacuoles using a human cerebral microvascular endothelial cell line (hCMEC/D3) cell monolayer. Here, we used daunorubicin (DNR) as a microtubule-targeting agent with the ability to disaggregate pre-formed fibrils and prevent Tau fibrillization. An in vitro model was developed by culturing hCMEC/D3 cells on Transwell inserts in EBM-2 endothelial basal medium until the cells formed a monolayer. Next, nano-sized yeast vacuoles were loaded with DNR, and the signals inside and outside the hMEC/D3 cell monolayer were detected using the GloMax® Explorer fluorometer. DNR penetrated the cell monolayer and was regulated by endocytosis via receptor-mediated macropinocytosis on the surface of the cell. Confocal imaging showed a significant increase in intracellular DNR fluorescence when the cells were treated with the vacuole-encapsulated drug. These results indicate that the drug penetrated the hCMEC/D3 cell monolayer via encapsulation into the vacuoles. Overall, yeast-derived vacuoles are promising candidates as drug carriers to the brain.

Atrial Natriuretic Peptide Associated with Cardiovascular Diseases Inhibits Amyloid-β Aggregation via Cross-Seeding.

Both cardiovascular diseases (CVDs) and Alzheimer's disease (AD) share some common risk factors (e.g., age, obesity, oxidative stress, inflammation, hypertension) that contribute to their overlapping pathogenesis, indicating a "head-to-heart" pathological connection between CVDs and AD. To explore this potential connection at the protein level, we study the potential cross-seeding (heterotypic interactions) between CVD-associated atrial natriuretic peptide (ANP) and AD-associated β-amyloid (Aβ). Collective aggregation and cell assays demonstrate the cross-seeding of ANP with different Aβ species including monomers, oligomers, and fibrils with high binding affinity (KD = 1.234-1.797 μM) in a dose-dependent manner. Such ANP-induced cross-seeding also modifies the Aβ aggregation pathway, fibril morphology, and cell deposition pattern by inhibiting Aβ fibrillization from small aggregates, disassembling preformed Aβ fibrils, and alleviating Aβ-associated cytotoxicity. Finally, using transgenic C. elegans worms that express the human muscle-specific Aβ1-42, ANP can also effectively delay Aβ-induced worm paralysis, decrease Aβ plaques in worm brains, and reduce reactive oxygen species (ROS) production, confirming its in vivo inhibition ability to prevent neurodevelopmental toxicity in worms. This work discovers not only a new cross-seeding system between the two disease-related proteins but also a new finding that ANP possesses a new biological function as an Aβ inhibitor in the nonaggregated state.

Molecular Mechanism of Cyanidin-3-O-Glucoside Disassembling Aβ Fibril In Silico

The deposition of β-amyloid (Aβ) in the brain leads to neurotoxic effects and subsequent Alzheimer's disease (AD). While AD is becoming more and more prevalent in modern society, therapeutic efforts targeting Aβ could be a promising solution. Currently, two natural products are reported to disintegrate preformed Aβ fibril in vitro. Meanwhile, the chemical driving force behind this phenomenon remains unknown. Taking cyanidin-3-O-glucoside (Cy-3G) as an example, here we studied its interaction with different Aβ polymorphs in silico. Negative charges on different Aβ polymorphs draw the interaction with the flavylium cation on Cy-3G. Our results show that Aβ in a single peptide form in solution exposed more hydrophobic solvent accessible surface area than its fibril structure (per protomer), and Cy-3G interacts more intensively with the single peptide form than fibril as indicated by more hydrogen bonding formed and more amino acid residues involved in their hydrophobic interactions. Thus, the single Aβ peptide aggregation into fibril and fibril dissociation into single peptide equilibrium could be disturbed by the preferential binding of Cy-3G to the monomeric Aβ peptide, which leads to the disassembly of the pathogenic Aβ fibril. This study offers a novel perspective of Cy-3G alleviated AD syndrome beyond its dogmatic antioxidant activity.

Aggregation of alpha-synuclein in enteric neurons does not impact function in vitro

Recent evidence implicates a gut-first pathogenesis in the enteric nervous system (ENS) within a portion of PD patients, yet in vitro investigations have primarily focused on the central nervous system. Here, the preformed fibril (PFF) PD model is applied with co-administered groups of butyrate and lipopolysaccharide to model the effects of the local gut microbiome. Significant PFF uptake and retention occur in isolated rat enteric neurons compared to untreated controls resulting in increasing immunostained aggregate conformation-specific, alpha-synuclein (a-Syn) average intensity between 6 µg PFF and untreated controls. Cortical neurons significantly retain PFFs with an increase in aggregated a-Syn average intensity within all dosages. Differences in growth cone morphology but not dynamics in PFF-treated ENS cultures occur. Electrophysiological recordings via a microelectrode array (MEA) indicate no overall difference in spontaneous spike rate. However, only untreated controls respond to PD-relevant dopamine stimulus, while 1 µg PFF and control populations respond to stimulus with ENS-abundant acetylcholine. Finally, no differences in substance P levels—correlated with PD and neurodegeneration—are observed. Overall, these findings suggest the ENS retains PFF dosage absent acute loss in function, however, does experience changes in growth cone morphology and dopamine-stimulated activity.

Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease

Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.

A Combined α-Synuclein/Fibril (SynFib) Model of Parkinson-Like Synucleinopathy Targeting the Nigrostriatal Dopamine System.

Injections of pre-formed α-synuclein fibrils (PFFs) or overexpression of α-synuclein using AAV vectors are commonly used as models of Parkinson-like synucleinopathy in rats and mice. In the modified method reviewed here, the "SynFib" model, the PFFs and the AAV vector are administered together unilaterally into the substantia nigra. This approach combines the key features of these two models, i.e., the generation of toxic α-synuclein aggregates and Lewy body-like inclusions, in combination with the increased vulnerability caused by increased cellular levels of α-synuclein. The combined AAV/PFF delivery offers several advantages over the standard PFF model due to the enhanced and accelerated α-synuclein pathology and microglial response induced by the PFF seeds in the presence of an elevated α-synuclein level. Injection of the AAV/PFF mixture into the substantia nigra makes it possible to target a larger proportion of the nigral dopamine neurons and obtain a level of dopamine cell loss (>60%) needed to induce significant impairments in drug-induced and spontaneous motor tests. The SynFib model shares attractive features of the standard 6-OHDA lesion model: a single unilateral stereotaxic intervention; pathology and cell loss developing over a short time span; and the possibility to monitor the degenerative changes using tests of motor behavior.