Mapping the lipidome in mitochondria-associated membranes (MAMs) in an in vitro model of Alzheimer's disease.

The disruption of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) plays a relevant role in Alzheimer's disease (AD). MAMs have been implicated in neuronal dysfunction and death since it is associated with impairment of functions regulated in this subcellular domain, including lipid synthesis and trafficking, mitochondria dysfunction, ER stress-induced unfolded protein response (UPR), apoptosis, and inflammation. Since MAMs play an important role in lipid metabolism, in this study we characterized and investigated the lipidome alterations at MAMs in comparison with other subcellular fractions, namely microsomes and mitochondria, using an in vitro model of AD, namely the mouse neuroblastoma cell line (N2A) over-expressing the APP familial Swedish mutation (APPswe) and the respective control (WT) cells. Phospholipids (PLs) and fatty acids (FAs) were isolated from the different subcellular fractions and analyzed by HILIC-LC-MS/MS and GC-MS, respectively. In this in vitro AD model, we observed a down-regulation in relative abundance of some phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) species with PUFA and few PC with saturated and long-chain FA. We also found an up-regulation of CL, and antioxidant alkyl acyl PL. Moreover, multivariate analysis indicated that each organelle has a specific lipid profile adaptation in N2A APPswe cells. In the FAs profile, we found an up-regulation of C16:0 in all subcellular fractions, a decrease of C18:0 levels in total fraction (TF) and microsomes fraction, and a down-regulation of 9-C18:1 was also found in mitochondria fraction in the AD model. Together, these results suggest that the over-expression of the familial APP Swedish mutation affects lipid homeostasis in MAMs and other subcellular fractions and supports the important role of lipids in AD physiopathology.

Restorative Effect of Microalgae Nannochloropsis oceanica Lipid Extract on Phospholipid Metabolism in Keratinocytes Exposed to UVB Radiation.

Ultraviolet B (UVB) radiation induces oxidative stress in skin cells, generating reactive oxygen species (ROS) and perturbing enzyme-mediated metabolism. This disruption is evidenced with elevated concentrations of metabolites that play important roles in the modulation of redox homeostasis and inflammatory responses. Thus, this research sought to determine the impacts of the lipid extract derived from the Nannochloropsis oceanica microalgae on phospholipid metabolic processes in keratinocytes subjected to UVB exposure. UVB-irradiated keratinocytes were treated with the microalgae extract. Subsequently, analyses were performed on cell lysates to ascertain the levels of phospholipid/free fatty acids (GC-FID), lipid peroxidation byproducts (GC-MS), and endocannabinoids/eicosanoids (LC-MS), as well as to measure the enzymatic activities linked with phospholipid metabolism, receptor expression, and total antioxidant status (spectrophotometric methods). The extract from N. oceanica microalgae, by diminishing the activities of enzymes involved in the synthesis of endocannabinoids and eicosanoids (PLA2/COX1/2/LOX), augmented the concentrations of anti-inflammatory and antioxidant polyunsaturated fatty acids (PUFAs), namely DHA and EPA. These concentrations are typically diminished due to UVB irradiation. As a consequence, there was a marked reduction in the levels of pro-inflammatory arachidonic acid (AA) and associated pro-inflammatory eicosanoids and endocannabinoids, as well as the expression of CB1/TRPV1 receptors. The microalgal extract also mitigated the increase in lipid peroxidation byproducts, specifically MDA in non-irradiated samples and 10-F4t-NeuroP in both control and post-UVB exposure. These findings indicate that the lipid extract derived from N. oceanica, by mitigating the deleterious impacts of UVB radiation on keratinocyte phospholipids, assumed a pivotal role in reinstating intracellular metabolic equilibrium.

Evaluation of Lipid Extracts from the Marine Fungi Emericellopsis cladophorae and Zalerion maritima as a Source of Anti-Inflammatory, Antioxidant and Antibacterial Compounds.

Marine environments occupy more than 70% of the earth's surface, integrating very diverse habitats with specific characteristics. This heterogeneity of environments is reflected in the biochemical composition of the organisms that inhabit them. Marine organisms are a source of bioactive compounds, being increasingly studied due to their health-beneficial properties, such as antioxidant, anti-inflammatory, antibacterial, antiviral, or anticancer. In the last decades, marine fungi have stood out for their potential to produce compounds with therapeutic properties. The objective of this study was to determine the fatty acid profile of isolates from the fungi Emericellopsis cladophorae and Zalerion maritima and assess the anti-inflammatory, antioxidant, and antibacterial potential of their lipid extracts. The analysis of the fatty acid profile, using GC-MS, showed that E. cladophorae and Z. maritima possess high contents of polyunsaturated fatty acids, 50% and 34%, respectively, including the omega-3 fatty acid 18:3 n-3. Emericellopsis cladophorae and Z. maritima lipid extracts showed anti-inflammatory activity expressed by the capacity of their COX-2 inhibition which was 92% and 88% of inhibition at 200 µg lipid mL-1, respectively. Emericellopsis cladophorae lipid extracts showed a high percentage of inhibition of COX -2 activity even at low concentrations of lipids (54% of inhibition using 20 µg lipid mL-1), while a dose-dependent behaviour was observed in Z. maritima. The antioxidant activity assays of total lipid extracts demonstrated that the lipid extract from E. cladophorae did not show antioxidant activity, while Z. maritima gave an IC20 value of 116.6 ± 6.2 µg mL-1 equivalent to 92.1 ± 4.8 µmol Trolox g-1 of lipid extract in the DPPH• assay, and 101.3 ± 14.4 µg mL-1 equivalent to 106.6 ± 14.8 µmol Trolox g-1 of lipid extract in the ABTS•+ assay. The lipid extract of both fungal species did not show antibacterial properties at the concentrations tested. This study is the first step in the biochemical characterization of these marine organisms and demonstrates the bioactive potential of lipid extracts from marine fungi for biotechnological applications.

Lipidomic Characterization and Antioxidant Activity of Macro- and Microalgae Blend.

Macro- and microalgae are currently recognized sources of lipids with great nutritional quality and attractive bioactivities for human health promotion and disease prevention. Due to the lipidomic diversity observed among algae species, giving rise to different nutritional and functional characteristics, the mixture of macro- and microalgae has the potential to present important synergistic effects resulting from the complementarity among algae. The aim of this work was to characterize for the first time the lipidome of a blend of macro- and microalgae and evaluate the antioxidant capacity of its lipid fraction. Fatty acids were profiled by GC-MS, the polar lipidome was identified by high resolution LC-MS, and ABTS+• and DPPH• assays were used to assess the antioxidant potential. The most abundant fatty acids were oleic (18:1 n-9), α-linolenic (18:3 n-3), and linoleic (18:2 n-6) acids. The lipid extract presented a beneficial n-6/n-3 ratio (0.98) and low values of atherogenic (0.41) and thrombogenic indices (0.27). The polar lipidome revealed 462 lipid species distributed by glycolipids, phospholipids, and betaine lipids, including some species bearing PUFA and a few with reported bioactivities. The lipid extract also showed antioxidant activity. Overall, the results are promising for the valorization of this blend for food, nutraceutical, and biotechnological applications.

Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition.

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.

Bioactivities of Lipid Extracts and Complex Lipids from Seaweeds: Current Knowledge and Future Prospects.

While complex lipids of seaweeds are known to display important phytochemical properties, their full potential is yet to be explored. This review summarizes the findings of a systematic survey of scientific publications spanning over the years 2000 to January 2021 retrieved from Web of Science (WoS) and Scopus databases to map the state of the art and identify knowledge gaps on the relationship between the complex lipids of seaweeds and their reported bioactivities. Eligible publications (270 in total) were classified in five categories according to the type of studies using seaweeds as raw biomass (category 1); studies using organic extracts (category 2); studies using organic extracts with identified complex lipids (category 3); studies of extracts enriched in isolated groups or classes of complex lipids (category 4); and studies of isolated complex lipids molecular species (category 5), organized by seaweed phyla and reported bioactivities. Studies that identified the molecular composition of these bioactive compounds in detail (29 in total) were selected and described according to their bioactivities (antitumor, anti-inflammatory, antimicrobial, and others). Overall, to date, the value for seaweeds in terms of health and wellness effects were found to be mostly based on empirical knowledge. Although lipids from seaweeds are little explored, the published work showed the potential of lipid extracts, fractions, and complex lipids from seaweeds as functional ingredients for the food and feed, cosmeceutical, and pharmaceutical industries. This knowledge will boost the use of the chemical diversity of seaweeds for innovative value-added products and new biotechnological applications.

Polar Lipids Composition, Antioxidant and Anti-Inflammatory Activities of the Atlantic Red Seaweed Grateloupia turuturu.

Grateloupia turuturu Yamada, 1941, is a red seaweed widely used for food in Japan and Korea which was recorded on the Atlantic Coast of Europe about twenty years ago. This seaweed presents eicosapentaenoic acid (EPA) and other polyunsaturated fatty acids (PUFAs) in its lipid fraction, a feature that sparked the interest on its potential applications. In seaweeds, PUFAs are mostly esterified to polar lipids, emerging as healthy phytochemicals. However, to date, these biomolecules are still unknown for G. turuturu. The present work aimed to identify the polar lipid profile of G. turuturu, using modern lipidomics approaches based on high performance liquid chromatography coupled to high resolution mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS). The health benefits of polar lipids were identified by health lipid indices and the assessment of antioxidant and anti-inflammatory activities. The polar lipids profile identified from G. turuturu included 205 lipid species distributed over glycolipids, phospholipids, betaine lipids and phosphosphingolipids, which featured a high number of lipid species with EPA and PUFAs. The nutritional value of G. turuturu has been shown by its protein content, fatty acyl composition and health lipid indices, thus confirming G. turuturu as an alternative source of protein and lipids. Some of the lipid species assigned were associated to biological activity, as polar lipid extracts showed antioxidant activity evidenced by free radical scavenging potential for the 2,2'-azino-bis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS●+) radical (IC50 ca. 130.4 µg mL-1) and for the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) radical (IC25 ca. 129.1 µg mL-1) and anti-inflammatory activity by inhibition of the COX-2 enzyme (IC50 ca. 33 µg mL-1). Both antioxidant and anti-inflammatory activities were detected using a low concentration of extracts. This integrative approach contributes to increase the knowledge of G. turuturu as a species capable of providing nutrients and bioactive molecules with potential applications in the nutraceutical, pharmaceutical and cosmeceutical industries.

Potential Anti-Obesity, Anti-Steatosis, and Anti-Inflammatory Properties of Extracts from the Microalgae Chlorella vulgaris and Chlorococcum amblystomatis under Different Growth Conditions.

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.

Valuing Bioactive Lipids from Green, Red and Brown Macroalgae from Aquaculture, to Foster Functionality and Biotechnological Applications.

Marine edible macroalgae have functional proprieties that might improve human health and wellbeing. Lipids represent a minor fraction of macroalgae, yet with major interest as main carriers of omega 3 polyunsaturated fatty acids and intrinsic bioactive properties. In this study, we used lipid extracts from the green macroalgae Ulva rigida and Codium tomentosum; the red Gracilaria gracilis,Palmaria palmata and Porphyra dioica; and the brown Fucus vesiculosus, produced in a land-based integrated multitrophic aquaculture (IMTA) system. We determined the lipid quality indices based on their fatty acid profiles and their bioactivities as putative antioxidant, anti-inflammatory and antiproliferative agents. The results reveal to be species-specific, namely U. rigida displayed the lowest atherogenicity and thrombogenicity indices. Palmaria palmata and F. vesiculosus lipid extracts displayed the lowest inhibitory concentration in the free radical scavenging antioxidant assays. Ulva rigida, C. tomentosum, P. palmata and P. dioica inhibited COX-2 activity by up to 80%, while P. dioica and P. palmata extracts showed the highest cytotoxic potential in the MDA-MB-231 breast cancer cells. This work enhances the valorization of macroalgae as functional foods and promising ingredients for sustainable and healthy diets and fosters new applications of high-valued algal biomass, in a species-specific context.

Lipidomic analysis of human primary hepatocytes following LXR activation with GW3965 identifies AGXT2L1 as a main target associated to changes in phosphatidylethanolamine.

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

New Insights into the Anti-Inflammatory and Antioxidant Properties of Nitrated Phospholipids.

Nitro-fatty acids (NO2 -FA) have been widely studied with regard to their identification, structural characterization, and biological actions. NO2 -FA could also be present endogenously esterified to phospholipids (PL), and NO2 -PL were already detected in cardiac mitochondria from diabetic rats and cardiomyoblasts subjected to starvation. However, the biological actions of NO2 -PL have been overlooked. In this study, we evaluate the antioxidant and anti-inflammatory potential of the nitrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) formed in vitro by incubation with NO2 BF4 , in a well-recognized mimetic model of nitroxidative stress. Nitrated POPC showed anti-radical ability to reduce both 2,2-diphenyl-1-picrylhydrazyl radical (DPPH• ) (IC20 = 225 ± 4 µg/mL; Trolox equivalent (TE) = 86 ± 6 µmol Trolox/g lipid) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS•+ ) (IC50 = 124 ± 2 µg/mL; TE = 152 ± 9 µmol Trolox/g lipid). Also, higher lag times were achieved in oxygen radical absorbance capacity (ORAC) assay for nitrated POPC, indicating a faster reaction with oxygen-derived radicals (TE = 1.03 ± 0.22 and TE = 1.30 ± 0.16 mmol Trolox/g lipid for nonmodified and nitrated POPC, respectively). Nitrated POPC showed the ability to inhibit lipid oxidation induced by the hydroxyl radical generated under Fenton reaction conditions, monitored by electrospray ionization (ESI) mass spectrometry (MS) using phosphatidylcholine (PtdCho) liposomes as a model of cell membrane. Nitrated POPC showed anti-inflammatory potential, as assessed by the inhibition of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages activated by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) in a well-described in vitro model of inflammation. Altogether, this study provides new clues regarding the antioxidant and anti-inflammatory potential of nitrated POPC, which should be explored in depth.

Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity.

The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC-MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 µg/mL and 12.9 µg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 µg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

Lipidomic Approaches towards Deciphering Glycolipids from Microalgae as a Reservoir of Bioactive Lipids.

In recent years, noteworthy research has been performed around lipids from microalgae. Among lipids, glycolipids (GLs) are quite abundant in microalgae and are considered an important source of fatty acids (FAs). GLs are rich in 16- and 18-carbon saturated and unsaturated fatty acids and often contain polyunsaturated fatty acids (PUFAs) like n-3 α-linolenic (ALA 18:3), eicosapentaenoic (EPA, 20:5) and docosahexaenoic (DHA, 22:6). GLs comprise three major classes: monogalactosyldiacyl glycerolipids (MGDGs), digalactosyl diacylglycerolipids (DGDGs) and sulfoquinovosyl diacylglycerolipids (SQDGs), whose composition in FA directly depends on the growth conditions. Some of these lipids are high value-added compounds with antitumoral, antimicrobial and anti-inflammatory activities and also with important nutritional significance. To fully explore GLs' bioactive properties it is necessary to fully characterize their structure and to understand the relation between the structure and their biological properties, which can be addressed using modern mass spectrometry (MS)-based lipidomic approaches. This review will focus on the up-to-date FA composition of GLs identified by MS-based lipidomics and their potential as phytochemicals.

Molecular insights into mitochondrial dysfunction in cancer-related muscle wasting.

Alterations in muscle mitochondrial bioenergetics during cancer cachexia were previously suggested; however, the underlying mechanisms are not known. So, the goal of this study was to evaluate mitochondrial phospholipid remodeling in cancer-related muscle wasting and its repercussions to respiratory chain activity and fiber susceptibility to apoptosis. An animal model of urothelial carcinoma induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and characterized by significant body weight loss due to skeletal muscle mass decrease was used. Morphological evidences of muscle atrophy were associated to decreased respiratory chain activity and increased expression of mitochondrial UCP3, which altogether highlight the lower ability of wasted muscle to produce ATP. Lipidomic analysis of isolated mitochondria revealed a significant decrease of phosphatidic acid, phosphatidylglycerol and cardiolipin in BBN mitochondria, counteracted by increased phosphatidylcholine levels. Besides the impact on membrane fluidity, this phospholipid remodeling seems to justify, at least in part, the lower oxidative phosphorylation activity observed in mitochondria from wasted muscle and their increased susceptibility to apoptosis. Curiously, no evidences of lipid peroxidation were observed but proteins from BBN mitochondria, particularly the metabolic ones, seem more prone to carbonylation with the consequent implications in mitochondria functionality. Overall, data suggest that bladder cancer negatively impacts skeletal muscle activity specifically by affecting mitochondrial phospholipid dynamics and its interaction with proteins, ultimately leading to the dysfunction of this organelle. The regulation of phospholipid biosynthetic pathways might be seen as potential therapeutic targets for the management of cancer-related muscle wasting.

Dietary curcumin counteracts extracellular transthyretin deposition: insights on the mechanism of amyloid inhibition.

The transthyretin amyloidoses (ATTR) are devastating diseases characterized by progressive neuropathy and/or cardiomyopathy for which novel therapeutic strategies are needed. We have recently shown that curcumin (diferuloylmethane), the major bioactive polyphenol of turmeric, strongly suppresses TTR fibril formation in vitro, either by stabilization of TTR tetramer or by generating nonfibrillar small intermediates that are innocuous to cultured neuronal cells. In the present study, we aim to assess the effect of curcumin on TTR amyloidogenesis in vivo, using a well characterized mouse model for familial amyloidotic polyneuropathy (FAP). Mice were given 2% (w/w) dietary curcumin or control diet for a six week period. Curcumin supplementation resulted in micromolar steady-state levels in plasma as determined by LC/MS/MS. We show that curcumin binds selectively to the TTR thyroxine-binding sites of the tetramer over all the other plasma proteins. The effect on plasma TTR stability was determined by isoelectric focusing (IEF) and curcumin was found to significantly increase TTR tetramer resistance to dissociation. Most importantly, immunohistochemistry (IHC) analysis of mice tissues demonstrated that curcumin reduced TTR load in as much as 70% and lowered cytotoxicity associated with TTR aggregation by decreasing activation of death receptor Fas/CD95, endoplasmic reticulum (ER) chaperone BiP and 3-nitrotyrosine in tissues. Taken together, our results highlight the potential use of curcumin as a lead molecule for the prevention and treatment of TTR amyloidosis.

Binding of epigallocatechin-3-gallate to transthyretin modulates its amyloidogenicity.

More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches for TTR amyloidosis that interfere with any step of the cascade of events leading to fibril formation have therapeutic potential. In this study we tested (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, as an inhibitor of TTR amyloid formation. We demonstrate that EGCG binds to TTR "in vitro" and "ex vivo" and that EGCG inhibits TTR aggregation "in vitro" and in a cell culture system. These findings together with the low toxicity of the compound raise the possibility of using EGCG in a therapeutic approach for familial amyloidotic polyneuropathy, the most frequent form of hereditary TTR amyloidosis.