The Surprising Nonlinear Effects of S100A9 Proteins in the Retina.

Age-related macular degeneration (AMD) is a complex disease in which inflammation is implicated as a key factor but the precise molecular mechanisms are poorly understood. AMD lesions contain an excess of the pro-inflammatory S100A9 protein, but its retinal significance was yet unexplored. S100A9 was shown to be intrinsically amyloidogenic in vitro and in vivo. Here, we hypothesized that the retinal effects of S100A9 are related to its supramolecular conformation. ARPE-19 cultures were treated with native dimeric and fibrillar S100A9 preparations, and cell viability was determined. Wild-type rats were treated intravitreally with the S100A9 solutions in the right eye and with the vehicle in the left. Retinal function was assessed longitudinally by electroretinography (ERG), comparing the amplitudes and configurations for each intervention. Native S100A9 had no impact on cellular viability in vitro or on the retinal function in vivo. Despite dispersed intracellular uptake, fibrillar S100A9 did not decrease ARPE-19 cell viability. In contrast, S100A9 fibrils impaired retinal function in vivo following intravitreal injection in rats. Intriguingly, low-dose fibrillar S100A9 induced contrasting in vivo effects, significantly increasing the ERG responses, particularly over 14 days postinjection. The retinal effects of S100A9 were further characterized by glial and microglial cell activation. We provide the first indication for the retinal effects of S100A9, showing that its fibrils inflicted retinal dysfunction and glial activation in vivo, while low dose of the same assemblies resulted in an unpredicted enhancement of the ERG amplitudes. These nonlinear responses highlight the consequences of self-assembly of S100A9 and provide insight into its pathophysiological and possibly physiological roles in the retina.

ApoE Isoforms Inhibit Amyloid Aggregation of Proinflammatory Protein S100A9.

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer's disease (AD). The amyloid co-aggregation of S100A9 with amyloid-ß (Aß) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.

Effect of Proinflammatory S100A9 Protein on Migration and Proliferation of Microglial Cells.

Alzheimer's disease (AD) is a multifactorial disease affecting aging population worldwide. Neuroinflammation became a focus of research as one of the major pathologic processes relating to the disease onset and progression. Proinflammatory S100A9 is the central culprit in the amyloid-neuroinflammatory cascade implicated in AD and other neurodegenerative diseases. We studied the effect of S100A9 on microglial BV-2 cell proliferation and migration. The responses of BV-2 cells to S100A9 stimulation were monitored in real-time using live cell microscopy, transcriptome sequencing, immunofluorescence staining, western blot analysis, and ELISA. We observed that a low dose of S100A9 promotes migration and proliferation of BV-2 cells. However, acute inflammatory condition (i.e., high S100A9 doses) causes diminished cell viability; it is uncovered that S100A9 activates TLR-4 and TLR-7 signaling pathways, leading to TNF-α and IL-6 expression, which affect BV-2 cell migration and proliferation in a concentration-dependent manner. Interestingly, the effects of S100A9 are not only inhibited by TNF-α and IL-6 antibodies. The addition of amyloid-ß (Aß) 1-40 peptide resumes the capacities of BV-2 cells to the level of low S100A9 concentrations. Based on these results, we conclude that in contrast to the beneficial effects of low S100A9 dose, high S100A9 concentration leads to impaired mobility and proliferation of immune cells, reflecting neurotoxicity at acute inflammatory conditions. However, the formation of Aß plaques may be a natural mechanism that rescues cells from the proinflammatory and cytotoxic effects of S100A9, especially considering that inflammation is one of the primary causes of AD.

The Stabilization of S100A9 Structure by Calcium Inhibits the Formation of Amyloid Fibrils.

The calcium-binding protein S100A9 is recognized as an important component of the brain neuroinflammatory response to the onset and development of neurodegenerative disease. S100A9 is intrinsically amyloidogenic and in vivo co-aggregates with amyloid-ß peptide and α-synuclein in Alzheimer's and Parkinson's diseases, respectively. It is widely accepted that calcium dyshomeostasis plays an important role in the onset and development of these diseases, and studies have shown that elevated levels of calcium limit the potential for S100A9 to adopt a fibrillar structure. The exact mechanism by which calcium exerts its influence on the aggregation process remains unclear. Here we demonstrate that despite S100A9 exhibiting α-helical secondary structure in the absence of calcium, the protein exhibits significant plasticity with interconversion between different conformational states occurring on the micro- to milli-second timescale. This plasticity allows the population of conformational states that favour the onset of fibril formation. Magic-angle spinning solid-state NMR studies of the resulting S100A9 fibrils reveal that the S100A9 adopts a single structurally well-defined rigid fibrillar core surrounded by a shell of approximately 15-20 mobile residues, a structure that persists even when fibrils are produced in the presence of calcium ions. These studies highlight how the dysregulation of metal ion concentrations can influence the conformational equilibria of this important neuroinflammatory protein to influence the rate and nature of the amyloid deposits formed.

Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism.

Amyloid fibrils-nanoscale fibrillar aggregates with high levels of order-are pathogenic in some today incurable human diseases; however, there are also many physiologically functioning amyloids in nature. The process of amyloid formation is typically nucleation-elongation-dependent, as exemplified by the pathogenic amyloid-ß peptide (Aß) that is associated with Alzheimer's disease. Spider silk, one of the toughest biomaterials, shares characteristics with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is an inherent property preserved by various spider silk proteins (spidroins). Both spidroins and Aß capped by spidroin N- and C-terminal domains, can assemble into macroscopic spider silk-like fibers that consist of straight nanofibrils parallel to the fiber axis as observed in native spider silk. While Aß forms amyloid nanofibrils through a nucleation-dependent pathway and exhibits strong cytotoxicity and seeding effects, spidroins spontaneously and rapidly form amyloid-like nanofibrils via a non-nucleation-dependent polymerization pathway that involves lateral packing of fibrils. Spidroin nanofibrils share amyloid-like properties but lack strong cytotoxicity and the ability to self-seed or cross-seed human amyloidogenic peptides. These results suggest that spidroins´ unique primary structures have evolved to allow functional properties of amyloid, and at the same time direct their fibrillization pathways to avoid formation of cytotoxic intermediates.

Proinflammatory S100A9 stimulates TLR4/NF-κB signaling pathways causing enhanced phagocytic capacity of microglial cells.

Alzheimer's disease (AD) is the main cause of dementia, affecting the increasingly aging population. Growing evidence indicates that neuro-inflammation plays crucial roles, e.g., the association between AD risk genes with innate immune functions. In this study, we demonstrate that moderate concentrations of pro-inflammatory cytokine S100A9 regulate immune response of BV2 microglial cells, i.e., the phagocytic capacity, reflected by elevated number of 1 µm diameter Dsred-stained latex beads in the cytoplasm. In contrast, at high S100A9 concentrations, both the viability and phagocytic capacity of BV2 cells drop substantially. Furthermore, it is uncovered that S100A9 affects phagocytosis of microglia via NF-κB signaling pathways. Application of related target-specific drugs, i.e., IKK and TLR4 inhibitors, effectively suppresses BV2 cells' immune responses. These results suggest that pro-inflammatory S100A9 activates microglial phagocytosis, and possibly contributes to the clearance of amyloidogenic species at the early stage of AD.

Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aß) peptides.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-ß (Aß) peptides, and Aß oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aß peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods-mainly spectroscopy and imaging techniques-to characterize Aß/Ni(II) interactions in vitro, for different Aß variants: Aß(1-40), Aß(1-40)(H6A, H13A, H14A), Aß(4-40), and Aß(1-42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aß monomers. Equimolar amounts of Ni(II) ions retard Aß aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aß binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aß dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aß monomers, while in a membrane-mimicking environment (SDS micelles) coil-coil helix interactions appear to be induced. For SDS-stabilized Aß oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aß aggregation processes that are involved in AD brain pathology.

Common Mechanisms Underlying α-Synuclein-Induced Mitochondrial Dysfunction in Parkinson's Disease.

Parkinson's disease (PD) is the most common neurological movement disorder characterized by the selective and irreversible loss of dopaminergic neurons in substantia nigra pars compacta resulting in dopamine deficiency in the striatum. While most cases are sporadic or environmental, about 10% of patients have a positive family history with a genetic cause. The misfolding and aggregation of α-synuclein (α-syn) as a casual factor in the pathogenesis of PD has been supported by a great deal of literature. Extensive studies of mechanisms underpinning degeneration of the dopaminergic neurons induced by α-syn dysfunction suggest a complex process that involves multiple pathways, including mitochondrial dysfunction and increased oxidative stress, impaired calcium homeostasis through membrane permeabilization, synaptic dysfunction, impairment of quality control systems, disruption of microtubule dynamics and axonal transport, endoplasmic reticulum/Golgi dysfunction, nucleus malfunction, and microglia activation leading to neuroinflammation. Among them mitochondrial dysfunction has been considered as the most primary target of α-syn-induced toxicity, leading to neuronal cell death in both sporadic and familial forms of PD. Despite reviewing many aspects of PD pathogenesis related to mitochondrial dysfunction, a systemic study on how α-syn malfunction/aggregation damages mitochondrial functionality and leads to neurodegeneration is missing in the literature. In this review, we give a detailed molecular overview of the proposed mechanisms by which α-syn, directly or indirectly, contributes to mitochondrial dysfunction. This may provide valuable insights for development of new therapeutic approaches in relation to PD. Antioxidant-based therapy as a potential strategy to protect mitochondria against oxidative damage, its challenges, and recent developments in the field are discussed.

S100A9 amyloid growth and S100A9 fibril-induced impairment of gamma oscillations in area CA3 of mouse hippocampus ex vivo is prevented by Bri2 BRICHOS.

The pro-inflammatory and highly amyloidogenic protein S100A9 is central to the amyloid-neuroinflammatory cascade in neurodegenerative diseases leading to cognitive impairment. Molecular chaperone activity of Bri2 BRICHOS has been demonstrated against a range of amyloidogenic polypeptides. Using a combination of thioflavin T fluorescence kinetic assay, atomic force microscopy and immuno electron microscopy we show here that recombinant Bri2 BRICHOS effectively inhibits S100A9 amyloid growth by capping amyloid fibrils. Using ex-vivo neuronal network electrophysiology in mouse brain slices we also show that both native S100A9 and amyloids of S100A9 disrupt cognition-relevant gamma oscillation power and rhythmicity in hippocampal area CA3 in a time- and protein conformation-dependent manner. Both effects were associated with Toll-like receptor 4 (TLR4) activation and were not observed upon TLR4 blockade. Importantly, S100A9 that had co-aggregated with Bri2 BRICHOS did not elicit degradation of gamma oscillations. Taken together, this work provides insights on the potential influence of S100A9 on cognitive dysfunction in Alzheimer's disease (AD) via gamma oscillation impairment from experimentally-induced gamma oscillations, and further highlights Bri2 BRICHOS as a chaperone against detrimental effects of amyloid self-assembly.

Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation.

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms.

Co-Aggregation of S100A9 with DOPA and Cyclen-Based Compounds Manifested in Amyloid Fibril Thickening without Altering Rates of Self-Assembly.

The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer's and Parkinson's, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson's treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases.

Human Polymerase δ-Interacting Protein 2 (PolDIP2) Inhibits the Formation of Human Tau Oligomers and Fibrils.

A central characteristic of Alzheimer's disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.

Polyoxometalates as Effective Nano-inhibitors of Amyloid Aggregation of Pro-inflammatory S100A9 Protein Involved in Neurodegenerative Diseases.

Pro-inflammatory and amyloidogenic S100A9 protein is central to the amyloid-neuroinflammatory cascade in neurodegenerative diseases. Polyoxometalates (POMs) constitute a diverse group of nanomaterials, which showed potency in amyloid inhibition. Here, we have demonstrated that two selected nanosized niobium POMs, Nb10 and TiNb9, can act as potent inhibitors of S100A9 amyloid assembly. Kinetics analysis based on ThT fluorescence experiments showed that addition of either Nb10 or TiNb9 reduces the S100A9 amyloid formation rate and amyloid quantity. Atomic force microscopy imaging demonstrated the complete absence of long S100A9 amyloid fibrils at increasing concentrations of either POM and the presence of only round-shaped and slightly elongated aggregates. Molecular dynamics simulation revealed that both Nb10 and TiNb9 bind to native S100A9 homo-dimer by forming ionic interactions with the positively charged Lys residue-rich patches on the protein surface. The acrylamide quenching of intrinsic fluorescence showed that POM binding does not perturb the Trp 88 environment. The far and near UV circular dichroism revealed no large-scale perturbation of S100A9 secondary and tertiary structures upon POM binding. These indicate that POM binding involves only local conformational changes in the binding sites. By using intrinsic and 8-anilino-1-naphthalene sulfonate fluorescence titration experiments, we found that POMs bind to S100A9 with a Kd of ca. 2.5 µM. We suggest that the region, including Lys 50 to Lys 54 and characterized by high amyloid propensity, could be the key sequences involved in S1009 amyloid self-assembly. The inhibition and complete hindering of S100A9 amyloid pathways may be used in the therapeutic applications targeting the amyloid-neuroinflammatory cascade in neurodegenerative diseases.

Natural Compound from Olive Oil Inhibits S100A9 Amyloid Formation and Cytotoxicity: Implications for Preventing Alzheimer's Disease.

Polyphenolic compounds in the Mediterranean diet have received increasing attention due to their protective properties in amyloid neurodegenerative and many other diseases. Here, we have demonstrated for the first time that polyphenol oleuropein aglycone (OleA), which is the most abundant compound in olive oil, has multiple potencies for the inhibition of amyloid self-assembly of pro-inflammatory protein S100A9 and the mitigation of the damaging effect of its amyloids on neuroblastoma SH-SY5Y cells. OleA directly interacts with both native and fibrillar S100A9 as shown by intrinsic fluorescence and molecular dynamic simulation. OleA prevents S100A9 amyloid oligomerization as shown using amyloid oligomer-specific antibodies and cross-ß-sheet formation detected by circular dichroism. It decreases the length of amyloid fibrils measured by atomic force microscopy (AFM) as well as reduces the effective rate of amyloid growth and the overall amyloid load as derived from the kinetic analysis of amyloid formation. OleA disintegrates already preformed fibrils of S100A9, converting them into nonfibrillar and nontoxic aggregates as revealed by amyloid thioflavin-T dye binding, AFM, and cytotoxicity assays. At the cellular level, OleA targets S100A9 amyloids already at the membranes as shown by immunofluorescence and fluorescence resonance energy transfer, significantly reducing the amyloid accumulation in GM1 ganglioside containing membrane rafts. OleA increases overall cell viability when neuroblastoma cells are subjected to the amyloid load and alleviates amyloid-induced intracellular rise of reactive oxidative species and free Ca2+. Since S100A9 is both a pro-inflammatory and amyloidogenic protein, OleA may effectively mitigate the pathological consequences of the S100A9-dependent amyloid-neuroinflammatory cascade as well as provide protection from neurodegeneration, if used within the Mediterranean diet as a potential preventive measure.

Elongation of wood fibers combines features of diffuse and tip growth.

Xylem fibers are highly elongated cells that are key constituents of wood, play major physiological roles in plants, comprise an important terrestrial carbon reservoir, and thus have enormous ecological and economic importance. As they develop, from fusiform initials, their bodies remain the same length while their tips elongate and intrude into intercellular spaces. To elucidate mechanisms of tip elongation, we studied the cell wall along the length of isolated, elongating aspen xylem fibers and used computer simulations to predict the forces driving the intercellular space formation required for their growth. We found pectin matrix epitopes (JIM5, LM7) concentrated at the tips where cellulose microfibrils have transverse orientation, and xyloglucan epitopes (CCRC-M89, CCRC-M58) in fiber bodies where microfibrils are disordered. These features are accompanied by changes in cell wall thickness, indicating that while the cell wall elongates strictly at the tips, it is deposited all over fibers. Computer modeling revealed that the intercellular space formation needed for intrusive growth may only require targeted release of cell adhesion, which allows turgor pressure in neighboring fiber cells to 'round' the cells creating spaces. These characteristics show that xylem fibers' elongation involves a distinct mechanism that combines features of both diffuse and tip growth.

Cholesterol-containing lipid nanodiscs promote an α-synuclein binding mode that accelerates oligomerization.

Dysregulation of the biosynthesis of cholesterol and other lipids has been implicated in many neurological diseases, including Parkinson's disease. Misfolding of α-synuclein (α-Syn), the main actor in Parkinson's disease, is associated with changes in a lipid environment. However, the exact molecular mechanisms underlying cholesterol effect on α-Syn binding to lipids as well as α-Syn oligomerization and fibrillation remain elusive, as does the relative importance of cholesterol compared to other factors. We probed the interactions and fibrillation behaviour of α-Syn using styrene-maleic acid nanodiscs, containing zwitterionic and anionic lipid model systems with and without cholesterol. Surface plasmon resonance and thioflavin T fluorescence assays were employed to monitor α-Syn binding, as well as fibrillation in the absence and presence of membrane models. 1 H-15 N-correlated NMR was used to monitor the fold of α-Syn in response to nanodisc binding, determining individual residue apparent affinities for the nanodisc-contained bilayers. The addition of cholesterol inhibited α-Syn interaction with lipid bilayers and, however, significantly promoted α-Syn fibrillation, with a more than a 20-fold reduction of lag times before fibrillation onset. When α-Syn bilayer interactions were analysed at an individual residue level by solution-state NMR, we observed two different effects of cholesterol. In nanodiscs made of DOPC, the addition of cholesterol modulated the NAC part of α-Syn, leading to stronger interaction of this region with the lipid bilayer. In contrast, in the nanodiscs comprising DOPC, DOPE and DOPG, the NAC part was mostly unaffected by the presence of cholesterol, while the binding of the N and the C termini was both inhibited.

Proinflammatory S100A9 Regulates Differentiation and Aggregation of Neural Stem Cells.

Inflammation is the primary pathological feature of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Proinflammatory molecules (e.g., S100A9) play important roles during the progression of the diseases by regulating behavior and fate of multiple cell types in the nervous system. Our earlier studies reveal that S100A9 is toxic to neurons, and its interaction with Aß peptides leads to the formation of large nontoxic amyloidogenic aggregates, suggesting a protective role of coaggregation with Aß amyloids. We herein demonstrate that S100A9 interacts with neural stem cells (NSCs) and causes NSC differentiation. In the brain of transgenic AD mouse models, we found large quantities of proinflammatory S100A9, which colocalizes with the differentiated NSCs. NSC sphere formation, which is a representative character of NSC stemness, is also substantially inhibited by S100A9. These results suggest that S100A9 is a representative marker for the inflammatory conditions in AD, and it promotes NSC differentiation. Intriguingly, in contrast to the death of both stem and differentiated NSCs caused by high S100A9 doses, S100A9 at a moderate concentration is toxic only to the early differentiated NSCs but not the stem cells. We therefore postulate that, at the early stage of AD, the expression of S100A9 leads to NSC differentiation, which remedies the neuron damage. The application of drugs, which help maintain NSC stemness (e.g., the platelet-derived growth factor, PDGF), may help overcome the acute inflammatory conditions and improve the efficacy of NSC transplantation therapy.

Surface-Directed Structural Transition of Amyloidogenic Aggregates and the Resulting Neurotoxicity.

The transition of amyloidogenic species into ordered structures (i.e., prefibrillar oligomers, protofibrils, mature fibrils, and amyloidogenic aggregates) is closely associated with many neurodegenerative disease pathologies. It is increasingly appreciated that the liquid-solid interface contributes to peptide aggregation under physiological conditions. However, much remains to be explored on the molecular mechanism of surface-directed amyloid formation. We herein demonstrate that physical environmental conditions (i.e., negatively charged surface) affect amyloid formation. Nontoxic amyloid aggregates quickly develop into intertwisting fibrils on a negatively charged mica surface. These fibrillar structures show significant cytotoxicity on both neuroblastoma cell-lines (SH-SY5Y) and primary neural stem cells. Our results suggest an alternative amyloid development pathway, following which Aß peptides form large amyloidogenic aggregates upon stimulation, and later transit into neurotoxic fibrillar structures while being trapped and aligned by a negatively charged surface. Conceivably, the interplay between chemical and physical environmental conditions plays important roles in the development of neurodegenerative diseases.

Templating S100A9 amyloids on Aß fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aß42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aß42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aß42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aß42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

Correction: HEWL interacts with dissipated oleic acid micelles, and decreases oleic acid cytotoxicity.

[This corrects the article DOI: 10.1371/journal.pone.0212648.].