Glycated albumin precipitation using aptamer conjugated magnetic nanoparticles.

To develop a strategy for the elimination of prefibrillar amyloid aggregates, a three-step non-modified DNA aptamer conjugation on silica-coated magnetic nanoparticles was carried out to achieve aptamer conjugated on MNP (Ap-SiMNP). Prefibrillar amyloid aggregates are generated under a diabetic condition which are prominently participated in developing diabetic complications. The binding properties of candidate DNA aptamer against serum albumin prefibrillar amyloid aggregates (AA20) were verified using electrophoretic mobility shift assay (EMSA) and surface plasmon resonance spectroscopy (SPR) analysis. The chloro-functionalized silica-coated MNPs were synthesized then a nano-targeting structure as aptamer conjugated on MNP (Ap-SiMNP) was constructed. Finally, Ap-SiMNP was verified for specific binding efficiency and AA20 removal using an external magnetic field. The candidate aptamer showed a high binding capacity at EMSA and SPR analysis (KD = 3.4 × 10─9 M) and successfully used to construct Ap-SiMNP. Here, we show a proof of concept for an efficient bio-scavenger as Ap-SiMNP to provide a promising opportunity to consider as a possible strategy to overcome some diabetic complications through specific binding/removal of toxic AA20 species.

Acute zoster plasma contains elevated amyloid, correlating with Aß42 and amylin levels, and is amyloidogenic.

Herpes zoster is associated with an increased dementia and neovascular macular degeneration risk and a decline in glycemic control in diabetes mellitus. Because amyloid is present and pathogenic in these diseases, we quantified amyloid, Aß40, Aß42, and amylin in 14 zoster and 10 control plasmas. Compared with controls, zoster plasma had significantly elevated amyloid that correlated with Aß42 and amylin levels and increased amyloid aggregation with addition of exogenous Aß42 or amylin. These results suggest that zoster plasma contains factor(s) that promotes aggregation of amyloidogenic peptides, potentially contributing to the toxic amyloid burden and explaining accelerated disease progression following zoster.

Carbonyl-protein content increases in brain and blood of female rats after chronic oxycodone treatment.

BACKGROUND: Opioids are the most effective drugs commonly prescribed to treat pain. Due to their addictive nature, opioid pain relievers are now second to marijuana, ahead of cocaine with respect to dependence. Ours and other studies suggest potential toxic effects of chronic opioid administration leading to neuronal degeneration. It has been suggested that protein carbonylation may represent a sensitive biomarker of cellular degeneration. To evaluate whether prolonged oxycodone administration is associated with accumulation of protein aggregates that may contribute to neuronal degeneration we measured protein carbonylation levels in brain and also in blood plasma of rats after 30-days of 15 mg/kg daily oxycodone administration. RESULTS: We observed a significant increase in the level of carbonylated proteins in rat brain cortex after 30-days of oxycodone treatment compare to that in water treated animals. Also, oxycodone treated rats demonstrated accumulation of insoluble carbonyl-protein aggregates in blood plasma. CONCLUSIONS: Our data suggests that tests detecting insoluble carbonyl-protein aggregates in blood may serve as an inexpensive and minimally invasive method to monitor neuronal degeneration in patients with a history of chronic opioid use. Such methods could be used to detect toxic side effects of other medications and monitor progression of aging and neurodegenerative diseases.

Unpacking the aggregation-oligomerization-fibrillization process of naturally-occurring hIAPP amyloid oligomers isolated directly from sera of children with obesity or diabetes mellitus.

The formation of amyloid oligomers and fibrils of the human islet amyloid polypeptide (hIAPP) has been linked with ß- cell failure and death which causes the onset, progression, and comorbidities of diabetes. We begin to unpack the aggregation-oligomerization-fibrillization process of these oligomers taken from sera of pediatric patients. The naturally occurring or real hIAPP (not synthetic) amyloid oligomers (RIAO) were successfully isolated, we demonstrated the presence of homo (dodecamers, hexamers, and trimers) and hetero-RIAO, as well as several biophysical characterizations which allow us to learn from the real phenomenon taking place. We found that the aggregation/oligomerization process is active in the sera and showed that it happens very fast. The RIAO can form fibers and react with anti-hIAPP and anti-amyloid oligomers antibodies. Our results opens the epistemic horizon and reveal real differences between the four groups (Controls vs obesity, T1DM or T2DM) accelerating the process of understanding and discovering novel and more efficient prevention, diagnostic, transmission and therapeutic pathways.

Serum amyloid A1 is involved in amyloid plaque aggregation and memory decline in amyloid beta abundant condition.

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by cognitive impairment, progressive neurodegeneration, and amyloid-ß (Aß) lesion. In the neuronal death and disease progression, inflammation is known to play an important role. Our previous study on acute-phase protein serum amyloid A1 (SAA1) overexpressed mice showed that the liver-derived SAA1 accumulated in the brain by crossing the brain blood barrier (BBB) and trigger the depressive-like behavior on mouse. Since SAA1 involved in immune responses in other diseases, we focused on the possibility that SAA1 may exacerbate the neuronal inflammation related to Alzheimer's disease. A APP/SAA overexpressed double transgenic mouse was generated using amyloid precursor protein overexpressed (APP)-c105 mice and SAA1 overexpressed mice to examine the function of SAA1 in Aß abundant condition. Comparisons between APP and APP/SAA1 transgenic mice showed that SAA1 exacerbated amyloid aggregation and glial activation; which lead to the memory decline. Behavior tests also supported this result. Overall, overexpression of SAA1 intensified the neuronal inflammation in amyloid abundant condition and causes the greater memory decline compared to APP mice, which only expresses Aß 1-42.

Amyloidogenic Nanoplaques in Blood Serum of Patients with Alzheimer's Disease Revealed by Time-Resolved Thioflavin T Fluorescence Intensity Fluctuation Analysis.

BACKGROUND: Biomarkers are central to current research on molecular mechanisms underlying Alzheimer's disease (AD). Their further development is of paramount importance for understanding pathophysiological processes that eventually lead to disease onset. Biomarkers are also crucial for early disease detection, before clinical manifestation, and for development of new disease modifying therapies. OBJECTIVE: The overall aim of this work is to develop a minimally invasive method for fast, ultra-sensitive and cost-effective detection of structurally modified peptide/protein self-assemblies in the peripheral blood and in other biological fluids. Specifically, we focus here on using this method to detect structured amyloidogenic oligomeric aggregates in the blood serum of apparently healthy individuals and patients in early AD stage, and measure their concentration and size. METHODS: Time-resolved detection of Thioflavin T (ThT) fluorescence intensity fluctuations in a sub-femtoliter observation volume element was used to identify in blood serum ThT-active structured amyloidogenic oligomeric aggregates, hereafter called nanoplaques, and measure with single-particle sensitivity their concentration and size. RESULTS: The concentration and size of structured amyloidogenic nanoplaques are significantly higher in the blood serum of individuals diagnosed with AD than in control subjects. CONCLUSION: A new method with the ultimate, single-particle sensitivity was successfully developed. The proposed approach neither relies on the use of immune-based probes, nor on the use of radiotracers, signal-amplification or protein separation techniques, and provides a minimally invasive test for fast and cost-effective early determination of structurally modified peptides/proteins in the peripheral blood, as shown here, but also in other biological fluids.

Cholesterol secosterol aldehyde adduction and aggregation of Cu,Zn-superoxide dismutase: Potential implications in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. While the fundamental causes of the disease are still unclear, the accumulation of Cu,Zn-superoxide dismutase (SOD1) immunoreactive aggregates is associated with familial ALS cases. Cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) are reported to contribute to neurodegenerative disease pathology by inducing protein modification and aggregation. Here we have investigated the presence of secosterol aldehydes in ALS SOD1-G93A rats and their capacity to induce SOD1 aggregation. Secosterol aldehydes were analyzed in blood plasma, spinal cord and motor cortex of ALS rats at the pre-symptomatic and symptomatic stages. Seco B was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression. In vitro experiments showed that both Seco A and Seco B induce the formation of high molecular weight (HMW) SOD1 aggregates with amorphous morphology. SOD1 adduction to ω-alkynyl-secosterols analyzed by click assay showed that modified proteins are only detected in the HMW region, indicating that secosterol adduction generates species highly prone to aggregate. Of note, SOD1-secosterol adducts containing up to five secosterol molecules were confirmed by MALDI-TOF analysis. Interestingly, mass spectrometry sequencing of SOD1 aggregates revealed preferential secosterol adduction to Lys residues located at the electrostatic loop (Lys 122, 128 and 136) and nearby the dimer interface (Lys 3 and 9). Altogether, our results show that secosterol aldehydes are increased in plasma of symptomatic ALS rats and represent a class of aldehydes that can potentially modify SOD1 enhancing its propensity to aggregate.

Exploring the Influence of Mutation on Transthyretin Aggregation in Heart Disease.

BACKGROUND: Transthyretin (TTR) is the transporter protein (55 kDa) that carries retinolbinding protein and Thyroxin (T4) in its functional tetramer form. Presence of the mutation in this protein (TTR) may lead to the dissociation of tetramers to monomer which unfolds and self-associates to form amyloid aggregates. Aggregation of this protein has been found to be associated with various lifethreatening disorders such as Coronary Artery Disease (CAD) which is the major cause of mortality and morbidity worldwide. METHODS: In the present communication, we have predicted mutation prone residues of TTR with the help of suspect server. Substitution (T139R with 95 score) occurring at the thyroid hormone binding site was selected for studying the mutational consequences on TTR. The effect of mutation on stability, functionality, aggregation and folding rate was analyzed by MuPro, DUET, SDM, SNAP2, Polyphen2, PASTA2.0, Aggrescan and Folding RaCe servers. The presence of TTR monomer in CAD plasma has been observed through Western blot analysis. RESULTS: T139R mutation may expose the buried regions of TTR protein which help in the self association and the increase in the stability may help in the TTR deposition. Structural analysis indicated that F and H strands of TTR are more prone to aggregation. Thus, T139R mutation might cause these residues to be aggregation prone and change in folding rate and validated TTR monomer in diseased cases by Western blot analysis. CONCLUSION: The observed results clearly indicated that the occurrence of this mutation is causing the impact on the structural and functional significance of TTR by interfering in the formation of tetramer. Thus, hindrance created to thyroxin transportation resulted in higher lipid levels in the blood that ultimately might promote the progression of the CAD.

Glyoxal administration induces formation of high molecular weight aggregates of hemoglobin exhibiting amyloidal nature in experimental rats: An in vivo study.

Glyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs). In the present study, we have investigated the effect of glyoxal on experimental rat hemoglobin in vivo after external administration of the α-dicarbonyl compound in animals. Gel electrophoretic profile of hemolysate collected from glyoxal-treated rats (32mg/kg body wt. dose) after one week exhibited the presence of some high molecular weight protein bands that were found to be absent for control, untreated rats. Mass spectrometric and absorption studies indicated that the bands represented hemoglobin. Further studies revealed that the fraction exhibited the presence of intermolecular cross ß-sheet structure. Thus glyoxal administration induces formation of high molecular weight aggregates of hemoglobin with amyloid characteristics in rats. Aggregated hemoglobin fraction was found to exhibit higher stability compared to glyoxal-untreated hemoglobin. As evident from mass spectrometric studies, glyoxal was found to modify Arg-30ß and Arg-31α of rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to induce amyloid-like aggregation of hemoglobin in rats. Considering the increased level of glyoxal in diabetes mellitus as well as its high reactivity, the above findings may be physiologically significant.

Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro.

C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer's amyloid ß (Aß) (1-40) and on that from D76N ß2-microglobulin (ß2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aß(1-40) and D76N ß2-m fibril formation in a Ca(2+)-independent manner. CRP and SAP interacted with fresh and aggregated Aß(1-40) and D76N ß2-m on the fibril-forming pathway. Interestingly, in the presence of Ca(2+), SAP first inhibited, then significantly accelerated D76N ß2-m fibril formation. Electron microscopically, the surface of the D76N ß2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed.