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.

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.

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.

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.

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.

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.

Pro-Inflammatory S100A9 Protein Aggregation Promoted by NCAM1 Peptide Constructs.

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aß peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self-assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.

Proinflammatory and amyloidogenic S100A9 induced by traumatic brain injury in mouse model.

Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer's and Parkinson's. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer's as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aß peptide deposits characteristic for Alzheimer's disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target.

S100A9-Driven Amyloid-Neuroinflammatory Cascade in Traumatic Brain Injury as a Precursor State for Alzheimer's Disease.

Pro-inflammatory and amyloidogenic S100A9 protein is an important contributor to Alzheimer's disease (AD) pathology. Traumatic brain injury (TBI) is viewed as a precursor state for AD. Here we have shown that S100A9-driven amyloid-neuroinflammatory cascade was initiated in TBI and may serve as a mechanistic link between TBI and AD. By analyzing the TBI and AD human brain tissues, we demonstrated that in post-TBI tissues S100A9, produced by neurons and microglia, becomes drastically abundant compared to Aß and contributes to both precursor-plaque formation and intracellular amyloid oligomerization. Conditions implicated in TBI, such as elevated S100A9 concentration, acidification and fever, provide strong positive feedback for S100A9 nucleation-dependent amyloid formation and delay in its proteinase clearance. Consequently, both intracellular and extracellular S100A9 oligomerization correlated with TBI secondary neuronal loss. Common morphology of TBI and AD plaques indicated their similar initiation around multiple aggregation centers. Importantly, in AD and TBI we found S100A9 plaques without Aß. S100A9 and Aß plaque pathology was significantly advanced in AD cases with TBI history at earlier age, signifying TBI as a risk factor. These new findings highlight the detrimental consequences of prolonged post-TBI neuroinflammation, which can sustain S100A9-driven amyloid-neurodegenerative cascade as a specific mechanism leading to AD development.

Co-aggregation of pro-inflammatory S100A9 with α-synuclein in Parkinson's disease: ex vivo and in vitro studies.

BACKGROUND: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of α-synuclein (α-syn) and S100A9 both in vitro and ex vivo in PD brain. METHODS: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and α-syn location and aggregation. In vitro studies revealing S100A9 and α-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods. RESULTS: Co-localized and co-aggregated S100A9 and α-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 µm outer diameter, with S100A9 and α-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and α-syn were shown to interact with each other via the α-syn C-terminus with an apparent dissociation constant of ca. 5 µM. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of α-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers. CONCLUSIONS: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving α-syn and S100A9 and leading to PD, similar to the effect of S100A9 and Aß co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

Intranasally Administered S100A9 Amyloids Induced Cellular Stress, Amyloid Seeding, and Behavioral Impairment in Aged Mice.

Amyloid formation and neuroinflammation are major features of Alzheimer's disease pathology. Proinflammatory mediator S100A9 was shown to act as a link between the amyloid and neuroinflammatory cascades in Alzheimer's disease, leading together with Aß to plaque formation, neuronal loss and memory impairment. In order to examine if S100A9 alone in its native and amyloid states can induce neuronal stress and memory impairment, we have administered S100A9 species intranasally to aged mice. Single and sequential immunohistochemistry and passive avoidance behavioral test were conducted to evaluate the consequences. Administered S100A9 species induced widespread cellular stress responses in cerebral structures, including frontal lobe, hippocampus and cerebellum. These were manifested by increased levels of S100A9, Bax, and to a lesser extent activated caspase-3 immunopositive cells. Upon administration of S100A9 fibrils, the amyloid oligomerization was observed in the brain tissues, which can further exacerbate cellular stress. The cellular stress responses correlated with significantly increased training and decreased retention latencies measured in the passive avoidance test for the S100A9 treated animal groups. Remarkably, the effect size in the behavioral tests was moderate already in the group treated with native S100A9, while the effect sizes were large in the groups administered S100A9 amyloid oligomers or fibrils. The findings demonstrate the brain susceptibility to neurotoxic damage of S100A9 species leading to behavioral and memory impairments. Intranasal administration of S100A9 species proved to be an effective method to study amyloid induced brain dysfunctions, and S100A9 itself may be postulated as a target to allay early stage neurodegenerative and neuroinflammatory processes.

Finke-Watzky Two-Step Nucleation-Autocatalysis Model of S100A9 Amyloid Formation: Protein Misfolding as "Nucleation" Event.

Quantitative kinetic analysis is critical for understanding amyloid mechanisms. Here we demonstrate the application of generic Finke-Watzky (F-W) two-step nucleation-autocatalytic growth model to the concentration-dependent amyloid kinetics of proinflammatory α-helical S100A9 protein at pH 7.4 and at 37 and 42 °C. The model is based on two pseudoelementary reaction steps applied without further analytical constraints, and its treatment of S100A9 amyloid self-assembly demonstrates that initial misfolding and ß-sheet formation, defined as "nucleation" step, spontaneously takes place within individual S100A9 molecules at higher rate than the subsequent fibrillar growth. The latter, described as an autocatalytic process, will proceed if misfolded amyloid-prone S100A9 is populated on a macroscopic time scale. Short lengths of S100A9 fibrils are consistent with the F-W model. The analysis of fibrillar length distribution by the Beker-Döring model demonstrates independently that such distribution is solely determined by slow fibril growth and there is no fragmentation or secondary pathways decreasing fibrillar length.

Immunochemical Detection of α-Synuclein Autoantibodies in Parkinson's Disease: Correlation between Plasma and Cerebrospinal Fluid Levels.

Autoantibodies to Parkinson's disease (PD) amyloidogenic protein, α-synuclein, were recognized as a prospective biomarker for early disease diagnostics, yet there is inconsistency in previous reports, potentially related to PD status. Therefore, plasma and cerebrospinal fluid (CSF) of the cross-sectional cohort of 60 individuals, including recently diagnosed PD patients with mild and moderate PD and age-matched controls, were examined by enzyme-linked immunosorbent assay (ELISA). Nonparametric statistics was used for data analysis. We found significantly elevated levels of α-synuclein autoantibodies in both plasma and CSF in mild PD compared to controls, followed by some decrease in moderate PD. Receiver operating characteristic and effect size analyses confirmed the diagnostic power of α-synuclein antibodies in both plasma and CSF. For the first time, we showed the correlation between plasma and CSF α-synuclein antibody levels for mild, moderate, and combined PD groups. This indicates the potentiality of α-synuclein antibodies as PD biomarker and the increased diagnostic power of their simultaneous analysis in plasma and CSF.