Prevents Amyloid Fibril Formation Research Articles

Arginine: a potential prophylactic supplement for transthyretin amyloidosis

Transthyretin (TTR) is an amyloidogenic protein associated with TTR amyloidosis (ATTR). Dissociation of TTR tetramers into TTR monomers causes TTR misfolding, resulting in amyloid fibril formation and triggering the onset of ATTR. Low-molecular-weight tetrameric TTR stabilizers are potential therapeutic agents to delay ATTR progression. However, the currently available drugs are expensive and cannot be used for prophylaxis. Therefore, in this study, we aimed to identify a prophylactic supplement that suppresses TTR amyloid formation. We investigated whether arginine, an amyloidogenic protein aggregation inhibitor, stabilizes tetrameric TTR, thereby preventing amyloid fibril formation. Immunoblotting showed that arginine mixed with wild-type TTR (TTRwt), ATTR V30M (Val30Met), and human serum samples reduced the amount of monomeric TTR but increased the tetramer/monomer ratio of TTR compared to those in the samples without arginine. Additionally, oral administration of arginine (5000 mg for five days) to healthy volunteers effectively increased the tetramer/monomer ratio of TTR in the serum. Thioflavin T test, a quantitative analysis method for amyloid fibril formation, showed that amyloid fibril formation was significantly suppressed with arginine compared to that without arginine. As arginine is a common supplement and non-toxic amino acid, it can be used as a promising prophylactic supplement to suppress amyloid fibril formation in ATTR.

Enhancing anti-amyloidogenic properties and antioxidant effects of Scutellaria baicalensis polyphenols through novel nanoparticle formation

Protein aggregation and oxidative stress have gained significant research attention due to their association with a group of diseases known as amyloidosis. Among the strategies developed to prevent amyloidosis, utilization of polyphenols stands out as one of the most commonly employed approaches. Scutellaria baicalensis is renowned as one of the foremost herbal sources of polyphenols. In this study, we employed a direct oxidative pyrolysis method for polymerizing S. baicalensis's polyphenols (SBPPs) after their extraction, resulting in the formation of novel SBPPs nanoparticles. Upon polymerization, SBPPs nanoparticles showed remarkable properties including heightened water solubility, increased surface area, modified surface functional groups, and enhanced stability. As a result of these diverse factors, there was a considerable enhancement in the anti-amyloidogenic properties and antioxidant effects of SBPPs nanoparticles compared to its bulk form. The fibrillation kinetics, AFM images, and cytotoxicity assays strongly indicate that SBPPs nanoparticles are more effective than SBPPs at preventing amyloid fibril formation and associated cell toxicity. Additionally, SBPPs nanoparticles demonstrated more effective prevention of reactive oxygen species (ROS) production. In conclusion, the use of SBPPs in nanoparticle form presents a promising strategy to enhance anti-amyloidogenic properties, mitigate oxidative stress, and offer potential therapeutic benefits for amyloidosis-related diseases.

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

Polyol and sugar osmolytes stabilize the molten globule state of α-lactalbumin and inhibit amyloid fibril formation

Protein misfolding and aggregation are associated with several human diseases such as Alzheimer's, Parkinson's, prion related disorders, type-II diabetes, etc. Different strategies using molecular chaperones, synthetic and naturally occurring small molecules, osmolytes, etc. have been used to prevent protein aggregation and amyloid fibril formation. In this study, we have used bovine α-lactalbumin at pH 1.6, 37 °C, and shaking conditions to promote amyloid fibril formation. Polyol and sugar osmolytes like glycerol, sorbitol, and trehalose have been used to inhibit the fibrillation of a number of proteins. In the present work, amyloid fibril formation of α-lactalbumin has been shown by ThT assay and AFM, while changes in the secondary structure during fibrillation has been followed by circular dichroism spectroscopy. Our results show that glycerol, sorbitol, and trehalose affect amyloid fibril formation of α-lactalbumin in a concentration-dependent manner. There is a delay in the lag phase of amyloid fibril formation in sorbitol and trehalose and complete inhibition in 6 M glycerol. Our results indicate that delay in the lag phase and inhibition of amyloid fibril formation are due to the stabilization of molten globule state by these osmolytes. At pH 1.6, the molten globule as well as the amyloid fibrils bind to ANS. However, when pH was shifted from 1.6 to 7, only the oligomeric and the fibrillar species bind to ANS due to refolding of molten globule state. The outcome of this study might be useful in designing small molecules which may stabilize the intermediate states, thus preventing amyloid fibril formation.

Construction of Self-Assembling Lipopeptide-Based Benign Nanovesicles to Prevent Amyloid Fibril Formation and Reduce Cytotoxicity of GxxxGxxxGxxxG Motif.

Alzheimer's disease, a progressive severe neurodegenerative disorder, has been until now incurable, in spite of serious efforts worldwide. We have designed self-assembled myristoyl-KPGPK lipopeptide-based biocompatible nanovesicles, which can inhibit amyloid fibrillation made by the transmembrane GxxxGxxxGxxxG motif of Aβ-protein and human myelin protein zero as well as reduce their neurotoxicity. Various spectroscopic and microscopic investigations illuminate that the lipopeptide-based nanovesicles dramatically inhibit random coil-to-β-sheet transformation of Aβ25-37 and human myelin protein zero protein precursor, which is the prerequisite of GxxxGxxxGxxxG motif-mediated fibril formation. Förster resonance energy transfer (FRET) assay using synthesized Cy-3 (FRET donor) and Cy-5 (FRET acceptor)-conjugated Aβ25-37 also exhibits that nanovesicles strongly inhibit the fibril formation of Aβ25-37. The mouse neuro-2a neuroblastoma cell line is used, which revealed the GxxxGxxxGxxxG-mediated cytotoxicity. However, the neurotoxicity has been diminished by co-incubating the GxxxGxxxGxxxG motif with the nanovesicles.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to β-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Anti-tuberculotic thionamide antibiotics show antioxidative and neuronal cytoprotective nature by inhibiting amyloid formation in human insulin and amyloid β-42

Oxidative stress, amyloid formation, impaired proteasomal degradation are hallmarks of neurodegenerative diseases like Alzheimer's (AD) and are targets for developing therapeutics against such diseases. Thionamide antibiotics are second-line anti-TB drugs. We studied the inhibitory action of two thionamide drugs, prothionamide and ethionamide, against amyloid formation using various biophysical techniques. We also studied the ability of these drugs to act as antioxidants. Here, we show that both drugs are potent inhibitors of in-vitro amyloid formation of human insulin and Aβ42 and protect cultured neuroblastoma cells against the toxic effects of amyloids. Various biophysical techniques like thioflavin-T binding assays, dynamic light-scattering (DLS), circular dichroism (CD), and transmission electron microscopy studies confirm that these drugs prevent amyloid fibril formation. CD and DLS measurements reveal that these drugs exert their anti-amyloid potency by stabilising the proteins in their native state. Their cytoprotective behaviour could be attributed to their antioxidant properties and their ability to inhibit in vitro lipid peroxidation, as confirmed by various antioxidant assays. This study reports for the first time a new facet of thionamide antibiotics as potential amyloid and oxidation inhibitors, with implications in reducing oxidative stress-related manifestations of AD, thereby opening avenues to be used as a therapeutic in AD.

In-vitro and in-silico investigation of protective mechanisms of crocin against E46K α-synuclein amyloid formation.

α-Synuclein is a presynaptic neuronal protein that is abundant in the human brain and is linked genetically and neuropathologically to Parkinson's disease (PD). The E46K mutation of the α-synuclein gene has been linked to autosomal dominant early-onset of PD. Crocin is a carotenoid chemical compound of saffron that has been shown antioxidant and neural protective activity. This study examined the effect of Crocin in preventing the amyloid fibril in the E46K α-synuclein, through in vitro studies and computational simulations. The result demonstrated that Crocin acts as a molecular chaperone to prevent amyloid fibril formation of E46K α-synuclein in a concentration-dependent manner. In fact, Crocin redirects E46K α-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway or at least reduce its aggregation tendency. Combined results from molecular dynamics and docking studies indicate that the inhibitory effect of the Crocin may be due to binding of the Crocin with the hydrophobic region (contact interface) of the α-synuclein which has the propensity to form amyloid aggregate. The results indicated Crocin can potentially bind to the C-terminal and mainly NAC (central hydrophobic region) domain of the E46K α-synuclein, and stabilizes the protein by masking the polymerization hotspot and consequently converting the protein into amyloid fibrils. These results support that Crocin is a effective inhibitor of E46K α-synuclein fibrillization and it could be considered as a potential therapeutic agent in the treatment of Parkinson disease.

Cyanine dyes derived inhibition of insulin fibrillization

The potential of novel cyanine dyes to inhibit the insulin amyloid formation was evaluated using thioflavin T fluorescence assay, quantum-chemical calculations, molecular docking and molecular dynamics simulations. According to the ability to suppress the insulin fibrillization under physiological conditions the examined compounds were found to follow the order: trimethines > pentamethines > monomethines > heptamethines. Of these, the trimethines 3-3 and 3-5, and pentamethines 5-3 and 5-9 almost completely prevented the protein aggregation by retarding both nucleation (except 3-3) and elongation processes. The quantum-chemical calculations revealed a complex relationship between the dye structure and its inhibitory effects. The molecular docking studies showed that most cyanines bind specifically to the L17 ladder of the B chain, located at the dry steric zipper of the insulin fibril protofilament, and form the stable complexes with the helices of the insulin monomer. The molecular dynamics simulations provided evidence for the increase of insulin helicity in the presence of cyanines. Collectively, the presented findings highlight two possible mechanisms by which cyanines can inhibit the insulin fibrillization: i) stabilization of the native protein structure followed by the retardation of the protein nucleation (all dyes); and ii) blocking the lateral extension of β-sheets via the dye-protein stacking interactions (3-3, 3-5, 5-3, 5-9). Overall, the obtained results may prove of importance for the design of small molecules capable of preventing amyloid fibril formation by insulin and other proteins.

Global Protein Stabilization Does Not Suffice to Prevent Amyloid Fibril Formation.

Mutations or cellular conditions that destabilize the native protein conformation promote the population of partially unfolded conformations, which in many cases assemble into insoluble amyloid fibrils, a process associated with multiple human pathologies. Therefore, stabilization of protein structures is seen as an efficient way to prevent misfolding and subsequent aggregation. This has been suggested to be the underlying reason why proteins living in harsh environments, such as the extracellular space, have evolved disulfide bonds. The effect of protein disulfides on the thermodynamics and kinetics of folding has been extensively studied, but much less is known on its effect on aggregation reactions. Here, we designed a single point mutation that introduces a disulfide bond in the all-α FF domain, a protein that, despite being devoid of preformed β-sheets, forms β-sheet-rich amyloid fibrils. The novel and unique covalent bond in the FF domain dramatically increases its thermodynamic stability and folding speed. Nevertheless, these optimized properties cannot counteract the inherent aggregation propensity of the protein, thus indicating that a high global protein stabilization does not suffice to prevent amyloid formation unless it contributes to hide from exposure the specific regions that nucleate the aggregation reaction.

A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.

Investigating the inhibitory effects of Seidlitzia rosmarinus extract on the amyloid fibril formation of ҡ-casein in the presence of dextran

Background: Formation of amyloid fibrils has been associated with different protein aggregation diseases. Many studies indicate that many proteins can be converted in vitro into amyloid structures. Isolated ҡ-casein (ҡ-CN) spontaneously forms amyloid fibrils under physiological conditions, so it is a convenient model for researching generic aspect of fibril formation. Materials and Methods: In this study the effect of aqueous extract of S. rosmarinus on the amyloid formation of ҡ-CN in the presence and absence of crowding agent, dextran, have been examined using Thioflavin T binding (ThT) assay, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy. Results: ThT binding assay showed that dextran increased the rate of amyloid fibril formation and S. rosmarinus extract retarded the amyloid fibril formation in κ-CN. In the presence of dextran however, the effect of S. rosmarinus extract on the amyloid formation of ҡ-CN was less than in its absence. Fluorescence spectroscopy results also demonstrated that dextran led to unfolding and increased the exposure hydrophobic area in ҡ-CN. S. rosmarinus extract efficiency decreased the exposure of hydrophobic regions in κ-CN, whereas in the presence of dextran this effect of extract was reduced. CD spectroscopy results exhibited that incubation of κ-CN with S. rosmarinus extract prevented a structural transition to a β-sheet. CD spectroscopy results also indicated that by adding dextran to reduced κ-CN β-sheet structures observed, which indicates structural change. S. rosmarinus extract however, prevented transition to β-sheet structural. Conclusion: In conclusion our finding suggests that S. rosmarinus extract prevents amyloid fibril formation in κ-CN, although this effect decreased in the presence of dextran.

The potential inhibitory effect of β-casein on the aggregation and deposition of Aβ1-42 fibrils in Alzheimer’s disease: insight from in-vitro and in-silico studies

Aβ1-40 and Aβ1-42 have been shown to be the main components of the amyloid plaques found in the extracellular environment of neurons in Alzheimer’s disease. β-Casein, a milk protein, has been shown to display a remarkable chaperone ability in preventing the aggregation of proteins. In this study, the ability of β-casein to suppress the amyloid fibril formation of Aβ1-42 has been examined through in vitro studies and molecular docking simulation. The results demonstrate the inhibitory effect of β-casein on fibril formation in Aβ1-42, in a concentration dependent manner, suggesting that the chaperone binds to the Aβ1-42 and prevents amyloid fibril formation. Molecular docking results show that the inhibitory effect of the β-casein may be due to binding of the chaperone with the aggregation-prone region of the Aβ1-42 mainly via hydrophobic interactions. β-Casein probably binds to the CHC and C-terminal domain of the Aβ1-42, and stabilizes proteins by inhibiting the conversion of monomeric Aβ1-42 into fibrils. Thus our data suggests that the hydrophobic interactions between β-casein and Aβ1-42 play an important role in the burial of the hydrophobic part of the Aβ1-42. This means that β-casein maybe considered for use in preventing amyloid fibril formation in degenerative diseases such as Alzheimer.

Inhibitory Role of β-Casein on the α-Synuclein Aggregation Associated with Parkinson’s Disease In Vitro

Large soluble oligomeric species are observed as probable intermediates during fibril formation in aggregations of Parkinson’s disease (PD). Fibrillar deposits occur in PD. Amyloid forms α-Synuclein is one of the main compounds aggregations. β-Casein, a member of the Casein family, has been demonstrated to inhibit α-Synuclein aggregation by chaperone-like activity. In this study, we investigated the effect of chaperone activity of β-Casein in preventing the aggregation of α-Synuclein protein. We have examined the effect of β-Casein in preventing α-Synuclein aggregation by using from Thioflavin T-binding assay, transmission electron microscopy, ANS-binding assay, circular dichroism spectroscopy and FTIR spectroscopy. Results from the ThT binding assay demonstrated an increase in the ThT fluorescence intensity of α-Synuclein incubated in absence of β-Casein but in its presence fluorescence intensity is decreased. Electron microscopy data also indicated that β-Casein decreased the aggregation content of α-Synuclein. ANS results also showed that β-Casein significantly decreased the the hydrophobic area in α-Synuclein incubated. Circular dichroism spectroscopy (CD) results also showed that β-sheet structures of α-Synuclein incubated change to structural α-helical and β-turn in presence of β-Casein. FTIR spectroscopy indicates the presence of β-sheet structures in α-Synuclein incubated in absence of β-Casein and β-sheet structures decreased in its presence. Thus, our results suggest that in vitro, β-Casein interacts with α-Synuclein fibrils, changes the α-Synuclein structure and prevents amyloid fibril formation. This means that β-Casein could be essential for therapies inhibiting aggregation and to be an important therapeutic drug against PD.

Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel

Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloid fibrillogenesis. It is modelled by exposure of the protein to non-physiological low pH in vitro and is inhibited by small molecule compounds, such as the drug tafamidis. We have recently identified a new mechano-enzymatic pathway of TTR fibrillogenesis in vitro, catalysed by selective proteolytic cleavage, which produces a high yield of genuine amyloid fibrils. This pathway is efficiently inhibited only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high affinity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here we show that TTR fibrillogenesis by the mechano-enzymatic pathway is indeed more potently inhibited by tolcapone than by tafamidis but neither, even in large molar excess, completely prevents amyloid fibril formation. In contrast, mds84, the prototype of our previously reported bivalent ligand TTR ‘superstabiliser’ family, is notably more potent than the monovalent ligands and we show here that this apparently reflects the critical additional interactions of its linker within the TTR central channel. Our findings have major implications for therapeutic approaches in TTR amyloidosis.

Inhibitory Effect of β-Casein on the Amyloid Fibril Formation of Aβ1–40 Associated with Alzheimer’s Disease

Alzheimer’s disease is associated with the fibril formation of β-amyloid peptide in extracellular plaque. β-Casein is a milk protein that has shown a remarkable ability to stabilize proteins by inhibiting their protein aggregation and precipitation. The aim of this study was to test in vitro the ability of β-casein to bind the Aβ1–40, change the structure and inhibit the formation of amyloid fibrils in Aβ1–40. Results from the ThT binding assay indicated that incubation of Aβ1–40 with β-casein retarded amyloid fibril formation of Aβ1–40 in a concentration dependent manner such that at a ratio of 1:1 (w:w) led to a significant reduction in the amount of fluorescent intensity. The results from transmission electron microscopy (TEM) also showed that β-casein significantly reduced the number and size of the Aβ1–40 fibrils, suggesting that the chaperone bound to the Aβ1–40 fibrils and/or interacted with the fibrils in some way. ANS results also showed that β-casein significantly decreased the exposed hydrophobic surface in Aβ1–40. Following an ANS binding assay, CD spectroscopy results also showed that incubation of Aβ1–40 resulted in a structural transition to a β-sheet. In the presence of β-casein, however, α-helical conformation was observed which indicated stabilization of the protein. These results reveal the highly efficacious chaperone action of β-casein against amyloid fibril formation of Aβ1–40. These results suggest that in vitro, β-casein binds to the Aβ1–40 fibrils, alters the Aβ1–40 structure and prevents amyloid fibril formation. This approach may result in the identification of a chaperone mechanism for the treatment of neurological diseases.

Dissociation of a BRICHOS trimer into monomers leads to increased inhibitory effect on Aβ42 fibril formation

The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid β-peptide (Aβ) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer. We show here increased dissociation of the BRICHOS trimer into monomers, by addition of detergents or of bis-ANS, known to bind to the putative substrate-binding site, or by introducing a Ser to Arg mutation expected to interfere with trimer formation. This leads to increased capacity to delay Aβ42 fibril formation. Cross-linking of the BRICHOS trimer with glutaraldehyde, in contrast, renders it unable to affect Aβ42 fibril formation. Moreover, proSP-C BRICHOS expressed in HEK293 cells is mainly monomeric, as detected by proximity ligation assay. Finally, proteolytic cleavage of BRICHOS in a loop region that is cleaved during proSP-C biosynthesis results in increased capacity to delay Aβ42 fibril formation. These results indicate that modulation of the accessibility of the substrate-binding site is a means to regulate BRICHOS activity.

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

Hsp27 suppresses the Cu2+-induced amyloidogenicity, redox activity, and cytotoxicity of α-synuclein by metal ion stripping

Aberrant copper homeostasis and oxidative stress have critical roles in several neurodegenerative diseases. Expression of heat-shock protein 27 (Hsp27) is elevated under oxidative stress as well as upon treatment with Cu2+, and elevated levels of Hsp27 are found in the brains of patients with Alzheimer and Parkinson diseases. We demonstrate, using steady-state and time-resolved fluorescence spectroscopy as well as isothermal titration calorimetry studies, that Hsp27 binds Cu2+ with high affinity (Kd ~10−11 M). Treating IMR-32 human neuroblastoma cells with Cu2+ leads to upregulation of endogenous Hsp27. Further, overexpression of Hsp27 in IMR-32 human neuroblastoma cells confers cytoprotection against Cu2+-induced cell death. Hsp27 prevents the deleterious interaction of Cu2+ with α-synuclein, the protein involved in Parkinson disease and synucleinopathies. Hsp27 attenuates Cu2+- or Cu2+–α-synuclein-mediated generation of reactive oxygen species and confers cytoprotection on IMR-32 cells as well as on mouse primary neural precursor cells. Hsp27 prevents Cu2+–ascorbate or Cu2+–α-synuclein–ascorbate treatment-induced increase in mitochondrial superoxide level and mitochondrial disorganization in IMR-32 cells. Hsp27 dislodges the α-synuclein-bound Cu2+ and prevents the Cu2+-mediated amyloidogenesis of α-synuclein. Our findings that Hsp27 binds Cu2+ with high affinity leading to beneficial effects and that Hsp27 can dislodge Cu2+ from α-synuclein, preventing amyloid fibril formation, indicate potential therapeutic strategies for neurodegenerative diseases involving aberrant Cu2+ homeostasis.

Repurposing Diflunisal for Familial Amyloid Polyneuropathy

Familial amyloid polyneuropathy, a lethal genetic disease caused by aggregation of variant transthyretin, induces progressive peripheral nerve deficits and disability. Diflunisal, a nonsteroidal anti-inflammatory agent, stabilizes transthyretin tetramers and prevents amyloid fibril formation in vitro. To determine the effect of diflunisal on polyneuropathy progression in patients with familial amyloid polyneuropathy. International randomized, double-blind, placebo-controlled study conducted among 130 patients with familial amyloid polyneuropathy exhibiting clinically detectable peripheral or autonomic neuropathy at amyloid centers in Sweden (Umeå), Italy (Pavia), Japan (Matsumoto and Kumamoto), England (London), and the United States (Boston, Massachusetts; New York, New York; and Rochester, Minnesota) from 2006 through 2012. Participants were randomly assigned to receive diflunisal, 250 mg (n=64), or placebo (n=66) twice daily for 2 years. The primary end point, the difference in polyneuropathy progression between treatments, was measured by the Neuropathy Impairment Score plus 7 nerve tests (NIS+7) which ranges from 0 (no neurological deficits) to 270 points (no detectable peripheral nerve function). Secondary outcomes included a quality-of-life questionnaire (36-Item Short-Form Health Survey [SF-36]) and modified body mass index. Because of attrition, we used likelihood-based modeling and multiple imputation analysis of baseline to 2-year data. By multiple imputation, the NIS+7 score increased by 25.0 (95% CI, 18.4-31.6) points in the placebo group and by 8.7 (95% CI, 3.3-14.1) points in the diflunisal group, a difference of 16.3 points (95% CI, 8.1-24.5 points; P < .001). Mean SF-36 physical scores decreased by 4.9 (95% CI, -7.6 to -2.2) points in the placebo group and increased by 1.5 (95% CI, -0.8 to 3.7) points in the diflunisal group (P < .001). Mean SF-36 mental scores declined by 1.1 (95% CI, -4.3 to 2.0) points in the placebo group while increasing by 3.7 (95% CI, 1.0-6.4) points in the diflunisal group (P = .02). By responder analysis, 29.7% of the diflunisal group and 9.4% of the placebo group exhibited neurological stability at 2 years (<2-point increase in NIS+7 score; P = .007). Among patients with familial amyloid polyneuropathy, the use of diflunisal compared with placebo for 2 years reduced the rate of progression of neurological impairment and preserved quality of life. Although longer-term follow-up studies are needed, these findings suggest benefit of this treatment for familial amyloid polyneuropathy. clinicaltrials.gov Identifier: NCT00294671.