Macrophages and Natural Killers Degrade α-Synuclein Aggregates.

Amyloid oligomers and fibrils are protein aggregates that exert a high cell toxicity. Efficient degradation of these protein aggregates can minimize the spread and progression of neurodegeneration. In this study, we investigate the properties of natural killer (NK) cells and macrophages in the degradation of α-synuclein (α-Syn) aggregates grown in a lipid-free environment and in the presence of phosphatidylserine and cholesterol (PS/Cho), which are lipids that are directly associated with the onset and progression of Parkinson's disease. We found that both types of α-Syn aggregates were endocytosed by neurons, which caused strong damage to cell endosomes. Our results also indicated that PS/Cho vesicles drastically increased the toxicity of α-Syn fibrils formed in their presence compared to the toxicity of α-Syn aggregates grown in a lipid-free environment. Both NK cells and macrophages were able to degrade α-Syn and α-Syn/Cho monomers, oligomers, and fibrils. Quantitative analysis of protein degradation showed that macrophages demonstrated substantially more efficient internalization and degradation of amyloid aggregates in comparison to NK cells. We also found that amyloid aggregates induced the proliferation of macrophages and NK cells and significantly changed the expression of their cytokines and chemokines.

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

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

Concentration of Phosphatidylserine Influence Rates of Insulin Aggregation and Toxicity of Amyloid Aggregates In Vitro.

Phosphatidylserine (PS) is a negatively charged lipid that plays a critically important role in cell apoptosis. Under physiological conditions, PS is localized on the cytosolic side of plasma membranes via ATP-dependent flippase-mediated transport. A decrease in the ATP levels in the cell, which is taken place upon pathological processes, results in the increase in PS concentration on the exterior part of the cell membranes. PS on the outer membrane surfaces attracts and activates phagocytes, which trigger cell apoptosis. This programed irreversible cell death is observed upon the progressive neurodegeneration, a hallmark of numerous amyloid associated pathologies, such as diabetes type 2 and Alzheimer's disease. In this study, we investigate the extent to which the rates of protein aggregation, which occurs upon amyloid pathologies, can be altered by the concentration of PS in large unilamellar vesicles (LUVs). We found that with an increase in the concentration of PS from 20 to 40% relative to the concentration of phosphatidylcholine and phosphatidylethanolamine, the rate of insulin aggregation, protein linked to diabetes type 2, and injection amyloidosis drastically increased. Furthermore, the concentration of PS in LUVs determined the secondary structure of protein aggregates formed in their presence. We also found that these structurally different aggregates exerted distinctly different cell toxicities. These findings suggest that a substantial decrease in cell viability, which is likely to take place upon aging, results in the increase in the concentration of PS in the outer plasma membranes, where it triggers the irreversible self-assembly of amyloidogenic proteins, which, in turn, causes the progressive neurodegeneration.

Unsaturated fatty acids uniquely alter aggregation rate of α-synuclein and insulin and change the secondary structure and toxicity of amyloid aggregates formed in their presence.

Docosahexaenoic (DHA) and arachidonic acids (ARA) are omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs). These molecules constitute a substantial portion of phospholipids in plasma membranes. Therefore, both DHA and ARA are essential diet components. Once consumed, DHA and ARA can interact with a large variety of biomolecules, including proteins such as insulin and α-synuclein (α-Syn). Under pathological conditions known as injection amyloidosis and Parkinson's disease, these proteins aggregate forming amyloid oligomers and fibrils, toxic species that exert high cell toxicity. In this study, we investigate the role of DHA and ARA in the aggregation properties of α-Syn and insulin. We found that the presence of both DHA and ARA at the equimolar concentrations strongly accelerated aggregation rates of α-Syn and insulin. Furthermore, LCPUFAs substantially altered the secondary structure of protein aggregates, whereas no noticeable changes in the fibril morphology were observed. Nanoscale Infrared analysis of α-Syn and insulin fibrils grown in the presence of both DHA and ARA revealed the presence of LCPUFAs in these aggregates. We also found that such LCPUFAs-rich α-Syn and insulin fibrils exerted significantly greater toxicities compared to the aggregates grown in the LCPUFAs-free environment. These findings show that interactions between amyloid-associated proteins and LCPUFAs can be the underlying molecular cause of neurodegenerative diseases.

Elucidation of the Effect of Phospholipid Charge on the Rate of Insulin Aggregation and Structure and Toxicity of Amyloid Fibrils.

The plasma membrane is a dynamic structure that separates the cell interior from the extracellular space. The fluidity and plasticity of the membrane determines a large number of physiologically important processes ranging from cell division to signal transduction. In turn, membrane fluidity is determined by phospholipids that possess different charges, lengths, and saturation states of fatty acids. A growing body of evidence suggests that phospholipids may play an important role in the aggregation of misfolded proteins, which causes pathological conditions that lead to severe neurodegenerative diseases. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane: phosphatidylcholine and phosphatidylethanolamine, zwitterions: phosphatidylserine and phosphatidylglycerol, lipids that possess a negative charge, and cardiolipin that has double negative charge on its polar head. Our results show that both zwitterions strongly inhibit insulin aggregation, whereas negatively charged lipids accelerate fibril formation. We also found that in the equimolar presence of zwitterions insulin yields oligomers that exert significantly lower cell toxicity compared to fibrils that were grown in the lipid-free environment. Such aggregates were not formed in the presence of negatively charged lipids. Instead, long insulin fibrils that had strong cell toxicity were grown in the presence of such negatively charged lipids. However, our results showed no correlation between the charge of the lipid and secondary structure and toxicity of the aggregates formed in its presence. These findings show that the secondary structure and toxicity are determined by the chemical structure of the lipid rather than by the charge of the phospholipid polar head.

Protein-to-lipid ratio uniquely changes the rate of lysozyme aggregation but does not significantly alter toxicity of mature protein aggregates.

Irreversible aggregation of misfolded proteins is the underlying molecular cause of numerous pathologies, including diabetes type 2, Alzheimer's, and Parkinson's diseases. Such an abrupt protein aggregation results in the formation of small oligomers that can propagate into amyloid fibrils. A growing body of evidence suggests that protein aggregation can be uniquely altered by lipids. However, the role of the protein-to-lipid (P:L) ratio on the rate of protein aggregation, as well as the structure and toxicity of corresponding protein aggregates remains poorly understood. In this study, we investigate the role of the P:L ratio of five different phospho- and sphingolipids on the rate of lysozyme aggregation. We observed significantly different rates of lysozyme aggregation at 1:1, 1:5, and 1:10 P:L ratios of all analyzed lipids except phosphatidylcholine (PC). However, we found that at those P:L ratios, structurally and morphologically similar fibrils were formed. As a result, for all studies of lipids except PC, mature lysozyme aggregates exerted insignificantly different cell toxicity. These results demonstrate that the P:L ratio directly determines the rate of protein aggregation, however, has very little if any effect on the secondary structure of mature lysozyme aggregates. Furthermore, our results point to the lack of a direct relationship between the rate of protein aggregation, secondary structure, and toxicity of mature fibrils.

Lipids uniquely alter the secondary structure and toxicity of amyloid beta 1-42 aggregates.

Abrupt aggregation of amyloid ß1-42 (Aß) peptide is a hallmark of Alzheimer's disease (AD), a severe pathology that affects more than 44 million people worldwide. A growing body of evidence suggests that lipids can uniquely alter rates of Aß1-42 aggregation. However, it remains unclear whether lipids only alter rates of protein aggregation or also uniquely modify the secondary structure and toxicity of Aß1-42 oligomers and fibrils. In this study, we investigated the effect of phosphatidylcholine (PC), cardiolipin (CL), and cholesterol (Chol) on Aß1-42 aggregation. We found that PC, CL and Chol strongly accelerated the rate of fibril formation compared to the rate of Aß1-42 aggregation in the lipid-free environment. Furthermore, anionic CL enabled the strongest acceleration of Aß1-42 aggregation compared to zwitterionic PC and uncharged Chol. We also found that PC, CL and Chol uniquely altered the secondary structure of early-, middle- and late-stage Aß1-42 aggregates. Specifically, CL and Chol drastically increased the amount of parallel ß-sheet in Aß1-42 oligomers and fibrils grown in the presence of these lipids. This caused a significant increase in the toxicity of Aß : CL and Aß : Chol compared to the toxicity of Aß : PC and Aß1-42 aggregates formed in the lipid-free environment. These results demonstrate that toxicity of Aß aggregates correlates with the amount of their ß-sheet content, which, in turn, is determined by the chemical structure of lipids present at the stage of Aß1-42 aggregation.

Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18 days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion, NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.

Lipids uniquely alter rates of insulin aggregation and lower toxicity of amyloid aggregates.

Amyloid formation is a hallmark of many medical diseases including diabetes type 2, Alzheimer's and Parkinson diseases. Under these pathological conditions, misfolded proteins self-assemble forming oligomers and fibrils, structurally heterogeneous aggregates that exhibit a large variety of shapes and forms. A growing body of evidence points to drastic changes in the lipid profile in organs affected by amyloidogenic diseases. In this study, we investigated the extent to which individual phospho- and sphingolipids, as well as their mixtures can impact insulin aggregation. Our results show that lipids and their mixtures uniquely alter rates of insulin aggregation simultaneously changing the secondary structure of protein aggregates that are grown in their presence. These structurally different protein-lipid aggregates impact cell viability to different extent while using distinct mechanisms of toxicity. These findings suggest that irreversible changes in lipid profiles of organs may trigger formation of toxic protein species that in turn are responsible for the onset and progression of amyloidogenic diseases.

Charge of Phospholipids Determines the Rate of Lysozyme Aggregation but Not the Structure and Toxicity of Amyloid Aggregates.

Biophysical properties of plasma membranes are determined by a chemical structure of phospholipids, including saturation of fatty acids and charge of polar heads of these molecules. Phospholipids not only determine fluidity and plasticity of membranes but also play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane on the aggregation properties of the lysozyme. We found that the charge of phospholipids determines the aggregation rate of lysozyme and the morphology of the protein aggregates. However, the secondary structure and toxicity of these protein specimens are determined by the chemical nature rather than the charge of phospholipids. These findings show that the charge of phospholipids can be a key factor that determines the stability and aggregation mechanism of amyloidogenic proteins.

Lipids uniquely alter secondary structure and toxicity of lysozyme aggregates.

Abrupt aggregation of misfolded proteins is a hallmark of the large group of amyloid pathologies that include diabetes type 2, Alzheimer and Parkinson's diseases. Protein aggregation yields oligomers and fibrils, ß-sheet-rich structures that exert cell toxicity. Microscopic examination of amyloid deposits reveals the presence of lipids membranes, which suggests that lipids can be involved in the process of pathogenic protein assembly. In this study, we show that lipids can uniquely alter the aggregation rates of lysozyme, a protein that is associated with systemic amyloidosis. Specifically, cardiolipin (CL), ceramide (CER), and sphingomyelin (SM) accelerate, phosphatidylcholine (PC) strongly inhibits, whereas phosphatidylserine (PS) has no effect on the rate of protein aggregation. Furthermore, lipids uniquely alter the secondary structure of lysozyme aggregates. Furthermore, we found that lysozyme aggregates grown in the presence of CL, CER, SM, PS, and CL:PC mixtures exert significantly lower production of reactive oxygen species and mitochondrial dysfunction compared to lysozyme:PC aggregates and lysozyme fibrils grown in the lipid-free environment. These findings suggest that a change in the lipid composition of cell membranes, which is taken place upon neurodegeneration, may trigger the formation of toxic protein species that otherwise would not be formed.

Length and Unsaturation of Fatty Acids of Phosphatidic Acid Determines the Aggregation Rate of Insulin and Modifies the Structure and Toxicity of Insulin Aggregates.

Phosphatidic acid (PA) is a unique plasma membrane lipid that contains fatty acids (FAs) with different lengths and degrees of unsaturation. Under physiological conditions, PA acts as a second messenger regulating a wide variety of cellular processes. At the same time, the role of PA under pathological conditions, which are caused by an abrupt aggregation of amyloid proteins, remains unclear. In this study, we investigated the effect of PA with different lengths and unsaturation of FAs on insulin aggregation. We found that PA with C16:0 FAs strongly inhibited insulin aggregation, whereas PA with C18:0 FAs accelerated it. Furthermore, PA with unsaturated (C18:1) FAs made the insulin form extremely long and thick fibrils that were not observed for PAs with saturated FAs. We also found that the presence of PA with C16:0 FAs resulted in the formation of aggregates that exerted significantly lower cell toxicity compared to the aggregates formed in the presence of PAs with C18:0 and C18:1 FAs. These results suggest that PA may play a key role in neurodegeneration.

Amyloid aggregates exert cell toxicity causing irreversible damages in the endoplasmic reticulum.

Amyloid oligomers and fibrils are protein aggregates that cause an onset and progression of many neurodegenerative diseases, diabetes type 2 and systemic amyloidosis. Although a growing body of evidence shows that oligomers and fibrils trigger mitochondrial dysfunction simultaneously enhancing production of reactive oxygen species, exact mechanisms by which these protein aggregates exert their toxicities remain unclear. In this study, we used advanced microscopic and spectroscopic methods to examine topography and structure of insulin aggregates grown in the lipid-free environment, as well as in the presence of major classes of phospho- and sphingolipids. We also employed a set of molecular markers to determine the extent to which insulin aggregates induce a damage of cell endoplasmic reticulum (ER), an important cell organelle used for calcium storage, protein synthesis and folding. Our results show that insulin aggregates activate the expression of Activating Transcription Factor 6 (ATF6), a transmembrane protein that is involved in unfolded protein response (UPR) of the stressed ER. At the same time, two other ER transmembrane proteins, Inositol Requiring 1 (IRE1α) and eLF2a, the product of PKR-like ER kinase (PERK), exhibited very low expression levels. Furthermore, amyloid aggregates trigger an expression of the 78-kDa glucose-regulated protein GRP78, which is also involved in the UPR. We also observed UPR-induced expression of a proapoptotic transcription factor CHOP, which, in turn, regulates expression of caspase 3 kinase and BCL2 protein family members, including the ER localized Bax. These findings show that insulin oligomers and fibrils induce UPR-associated ER stress and ultimately fatal changes in cell homeostasis.

Lipids reverse supramolecular chirality and reduce toxicity of amyloid fibrils.

Abrupt aggregation of misfolded proteins is a hallmark of many medical pathologies including diabetes type 2, Alzheimer and Parkinson diseases. This results in the formation of amyloid fibrils, protein aggregates with distinct supramolecular chirality. A growing body of evidence suggests that lipids can alter rates of protein aggregation. In this study, we investigated whether lipids could alter the supramolecular chirality of amyloid fibrils. We found that if present at the stage of protein aggregation, phospho- and sphingolipids uniquely reversed supramolecular chirality of insulin and lysozyme fibrils. Furthermore, amyloid fibrils with opposite supramolecular chirality exerted distinctly different cell toxicity. Specifically, insulin and lysozyme fibrils with reversed supramolecular chirality were less toxic to cells than the aggregates with normal supramolecular chirality. These findings point on the important role of lipids and supramolecular chirality of amyloid fibrils in the onset and progression of amyloid diseases.

Characterization of Substrates and Surface-Enhancement in Atomic Force Microscopy Infrared Analysis of Amyloid Aggregates.

Atomic force microscopy infrared (AFM-IR) spectroscopy is an emerging analytical technique that can be used to probe the structural organization of specimens with nanometer spatial resolution. A growing body of evidence suggests that nanoscale structural analysis of very small (<10 nm) biological objects, such as viruses and amyloid aggregates, requires substrates that must fit strict criteria of low surface roughness and low IR background, simultaneously. In this study, we examine the suitability of a broad range of substrates commonly used in AFM and IR fields, and we determined that silicon, zinc sulfide, and calcium fluoride are the most ideal substrates for nanoscale imaging of amyloid oligomers, protein aggregates that are directly linked to the onset and progression of neurodegenerative diseases. Our data show that these substrates provide the lowest roughness and the lowest background in the 800-1800 cm-1 spectral window from all examined AFM and IR substrates. We also investigate a contribution of surface enhancement in AFM-IR by the direct comparison of signal intensities from oligomers located on silicon and gold-coated silicon surfaces. We found that metallization of such substrates provides a factor of ~7 enhancements to the IR signal and induces an equivalent enhancement of the sample background in the 950-1250 cm-1 spectral region.

The degree of unsaturation of fatty acids in phosphatidylserine alters the rate of insulin aggregation and the structure and toxicity of amyloid aggregates.

Phosphatidylserine (PS) in the plasma membrane plays an important role in cell signaling and apoptosis. Cell degeneration is also linked to numerous amyloid diseases, pathologies that are associated with aggregation of misfolded proteins. In this work, we examine the effect of both saturated PS (DMPS) and unsaturated PS (DOPS and POPS) on the aggregation properties of insulin, as well as the structure and toxicity of insulin aggregates formed in the presence of these phospholipids. We found that the degree of unsaturation of fatty acids in PS alters the rate of insulin aggregation. We also found that toxicity of insulin-DMPS aggregates is significantly lower than the toxicity of DOPS- and POPS-insulin fibrils, whereas all these lipid-containing aggregates exert lower cell toxicity than insulin fibrils grown in a lipid-free environment.

Unsaturation in the Fatty Acids of Phospholipids Drastically Alters the Structure and Toxicity of Insulin Aggregates Grown in Their Presence.

Lipid bilayers play an important role in the pathological assembly of amyloidogenic proteins and peptides. This assembly yields oligomers and fibrils, which are highly toxic protein aggregates. In this study, we investigated the role of saturation in fatty acids of two phospholipids that are present in cell membranes. We found that unsaturated cardiolipin (CL) drastically shortened the lag phase of insulin aggregation. Furthermore, structurally and morphologically different aggregates were formed in the presence of unsaturated CL vs saturated CL. These aggregates exerted drastically different cell toxicity. Both saturated and unsaturated phosphatidylcholine (PC) were able to inhibit insulin aggregation equally efficiently. Similar to CL, structurally different aggregates were formed in the presence of saturated and unsaturated PC. These aggregates exerted different cell toxicities. These results show that unsaturated phospholipids catalyze the formation of more toxic amyloid aggregates comparing to those formed in the presence of saturated lipids.

Nanoscale Structural Analysis of a Lipid-Driven Aggregation of Insulin.

Abrupt aggregation of misfolded proteins is a hallmark of a large number of severe pathologies, including diabetes types 1 and 2, Alzheimer, and Parkinson diseases. A growing body of evidence suggests that lipids can uniquely change rates of amyloid-associated proteins as well as modify the structure of formed oligomers and fibrils. In this study, we utilize atomic force microscopy infrared (AFM-IR) spectroscopy, also known as nano-IR spectroscopy, to examine the structure of individual insulin oligomers, protofilaments, and fibrils grown in the presence of phospholipids. Our findings show that AFM-IR spectra of insulin oligomers have strong signals of C-H and PO2- vibrations, which points on the presence of lipids in the oligomer structure. Furthermore, substantial shifts in lipid vibrations in AFM-IR spectra of the oligomers relative to the corresponding bands of pure lipids have been observed. This points on strong interactions between a lipid and a protein that are developed at the stage of the oligomer formation.