thioflavin-T Staining Research Articles

Investigation of serum amyloid a within animal species focusing on the 1-25 amino acid region

AA amyloidosis, characterized by the misfolding of serum amyloid A (SAA) protein, is the most common amyloid protein disorder across multiple species. SAA is a positive-acute phase protein synthesized by the liver in response to inflammation or stress, and it normally associates with high-density lipoprotein at its N-terminus. In this study, we focused on the 1-25 amino acid (aa) region of the complete 104 aa SAA sequence to examine the aggregation propensity of AA amyloid. A library comprising eight peptides from different species was assembled for analysis. To access the aggregation propensity of each peptide region, a bioinformatic study was conducted using the algorithm TANGO. Congo red (CR) binding assays, Thioflavin T (ThT) assays, and transmission electron microscopy (TEM) were utilized to evaluate whether the synthesized peptides formed amyloid-like fibrils. All synthetic SAA 1-25 congeners resulted in amyloid-like fibrils formation (per CR and/or ThT staining and TEM detection) at the exception of the ferret SAA1-25 fragment, which generated plaque-like materials by TEM. Ten residues were preserved among SAA 1-25 congeners resulting in amyloid-like fibrils, i.e. F6, E9, A10, G13, D16, M17, A20, Y21, D23, and M24. Amino acid residues highlighted by this study may have a role in increasing the propensity for amyloid-like fibril formation. This study put an emphasis on region 1-25 in the mechanism of SAA1 misfolding.

1759-P: Relation between Metabolic Remodeling in Pancreatic ß-Cells and Pancreas Histology in Autopsy Cases with Type 2 Diabetes

Background and Aims: Metabolic remodeling from oxidative phosphorylation to glycolysis via activation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) occurs in pancreatic β-cells of non-Asian individuals with type 2 diabetes (T2D), resulting in impaired β-cell function. To elucidate the underlying mechanism of metabolic remodeling, we investigated the relationship between PFKFB3 expression in β-cells and histological features of the pancreas in Japanese adults with T2D. Methods: Autopsy pancreatic sections from cadaveric donors with T2D and those without diabetes (ND) were stained with antibodies against PFKFB3, insulin, and glucagon. Ratios of PFKFB3-positive pancreatic β-cells and α-cells were compared between T2D and ND groups. Next, the relationship between PFKFB3 positivity in β-cells and histological features of the islets and pancreas were explored. We conducted thioflavin-T staining to identify amyloid deposition in islets of the T2D group and Azan staining was applied to evaluate the extent of fibrosis in ​​the pancreas. Results: In total, 36 cases (18 cases each from T2D and ND) were analyzed and 100 islets/case were evaluated. Mean ages of T2D and ND individuals were 70.3 ± 7.7 and 64.3 ± 12.8 years, and body mass indexes were 22.7 ± 4.4 and 23.3 ± 4.5 kg/m2, respectively. The PFKFB3-positive β-cell ratio was significantly higher in the T2D group (23.9 ± 16.1% vs 8.7 ± 6.3%, p < 0.05), whereas the PFKFB3-positive α-cell ratio did not differ between groups. A negative correlation between PFKFB3-positive β-cell ratio and pancreatic β-cell mass was found (ρ = −0.34, p < 0.05). In the T2D group, amyloid deposition inside the islets and pancreatic parenchymal fibrosis correlated with β-cell PFKFB3 positivity (ρ = 0.50 and ρ = 0.84; p < 0.05, respectively). Conclusion: Intracellular metabolic remodeling mediated by PFKFB3 occurred in Japanese adults with T2D and histological features of the pancreas were correlated with such changes. Disclosure R.Izumihara: None. H.Nomoto: Speaker's Bureau; Novo Nordisk, Sumitomo Pharma, Co., Ltd. K.Chiba: None. H.Kameda: None. A.Nakamura: Research Support; Abbott Japan Co., Ltd., Boehringer Ingelheim Japan, Inc., Daiichi Sankyo, Taisho Pharmaceutical Holdings Co., Ltd., Teijin Pharma Limited, Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation. H.Miyoshi: Research Support; Abbott, LifeScan Diabetes Institute, Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; AstraZeneca, Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Kowa Company, Ltd., MSD Life Science Foundation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Mitsubishi Tanabe Pharma Corporation. H.Mizukami: None. T.Atsumi: Speaker's Bureau; Eli Lilly Japan K.K., Kowa Company, Ltd. Funding Japan Society for the Promotion of Science (JP20281167); Japan Diabetes Society; MSD Life Science Foundation; Public Interest Incorporated Foundation

Controlling Amyloid Beta Peptide Aggregation and Toxicity by Protease-Stable Ligands.

Polymerization of soluble amyloid beta (Aβ) peptide into protease-stable insoluble fibrillary aggregates is a critical step in the pathogenesis of Alzheimer's disease (AD). The N-terminal (NT) hydrophobic central domain fragment 16KLVFF20 plays an important role in the formation and stabilization of β-sheets by self-recognition of the parent Aβ peptide, followed by aggregation of Aβ in the AD brain. Here, we analyze the effect of the NT region inducing β-sheet formation in the Aβ peptide by a single amino acid mutation in the native Aβ peptide fragment. We designed 14 hydrophobic peptides (NT-01 to NT-14) by a single mutation at 18Val by using hydrophobic residues leucine and proline in the natural Aβ peptide fragment (KLVFFAE) and analyzed its effect on the formation of Aβ aggregates. Among all these peptides, NT-02, NT-03, and NT-13 significantly affected the Aβ aggregate formation. When the NT peptides were coincubated with the Aβ peptide, a significant reduction in β-sheet formation and increment in random coil content of Aβ was seen, confirmed by circular dichroism spectroscopy and Fourier transform infrared spectroscopy, followed by the reduction of fibril formation measured by the thioflavin-T (ThT) binding assay. The aggregation inhibition was monitored by Congo red and ThT staining and electron microscopic examination. Moreover, the NT peptides protect the PC-12 differentiated neurons from Aβ-induced toxicity and apoptosis in vitro. Thus, manipulation of the Aβ secondary structure with protease-stable ligands that promote the random coil conformation may provide a tool to control the Aβ aggregates observed in AD patients.

Effects of Cu2+ on conformational change and aggregation of hPrP180-192 with a V180I mutation of the prion protein

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNμ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a β-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc.

Adenosine protects pancreatic beta cells against apoptosis induced by endoplasmic reticulum stress.

Chronic exposure to high glucose induces endoplasmic reticulum (ER) stress in pancreatic beta cells (PBCs). The previous evidence showed that adenosine modulate PBCs viability and insulin secretion. The aim of this study was to evaluate possible involvement of adenosine in protection of MIN6 β-cells from Tunicamycin (Tu)-induced ER stress. MIN6 cells were cotreated with Tu and different concentrations of adenosine. Cell viability, proliferation, and apoptosis were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), 5-bromo-2'-deoxyuridine (Brdu), and colony formation assays. Caspase-12 activity was assayed using the fluorometric method. Thioflavin T (ThT) staining was used for the evaluation of protein aggregation. Insulin secretion was evaluated using specific an ELISA kit. Ca2+ mobilization assayed using Fura2/AM probe. BIP, CHOP, XBP-1, and XBP-1s expression in both messenger RNA (mRNA) and protein levels were evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Bcl-2, p-eIF2α/eIF2α, and GADD34 levels also determined with Western blot analysis. Adenosine protected MIN6 cells against Tu-induced ER stress in a dose-dependent manner and increased their proliferation. Decreased caspase-12 activity and upregulated Bcl-2 protein may explain antiapoptotic effects of adenosine. ThT staining indicated an attenuated aggregation of misfolded proteins. Adenosine effectively increased insulin secretion in Tu-treated cells. BIP, CHOP, XBP1, and sXBP1 expression were decreased significantly in cotreated cells, indicating alleviation of ER stress. However, adenosine potentiated the expression of GADD34 and decreased p-eIF2α/eIF2α ratio. Adenosine increased cytosolic Ca 2+ levels, which may promote adenosine triphosphate (ATP) synthesis in mitochondria, helping ER to preserve protein hemostasis. Taken together, adenosine upregulated Bcl-2 and GADD34 to protect PBCs against Tu-induced apoptosis and increase Insulin secretion.

Bonsai Gelsolin Survives Heat Induced Denaturation by Forming \u03b2-Amyloids which Leach Out Functional Monomer

Here, we report that minimal functional gelsolin i.e. fragment 28–161 can display F-actin depolymerizing property even after heating the protein to 80 °C. Small angle X-ray scattering (SAXS) data analysis confirmed that under Ca2+-free conditions, 28–161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca2+, it forms dimers which proceed to non-characterizable aggregates. The dimeric association also explained the observed decrease in ellipticity in circular dichroism experiments with increase in temperature. Importantly, SAXS data based models correlated well with our crystal structure of dimeric state of 28–161. Characterization of higher order association by electron microscopy, Congo red and ThioflavinT staining assays further confirmed that only in absence of Ca2+ ions, heating transforms 28–161 into β-amyloids. Gel filtration and other experiments showed that β-amyloids keep leaching out the monomer, and the release rates could be enhanced by addition of L-Arg to the amyloids. F-actin depolymerization showed that addition of Ca2+ ions to released monomer initiated the depolymerization activity. Overall, we propose a way to compose a supramolecular assembly which releases functional protein in sustained manner which can be applied for varied potentially therapeutic interventions.

Royal Jelly Reduces Cholesterol Levels, Ameliorates Aβ Pathology and Enhances Neuronal Metabolic Activities in a Rabbit Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common form of dementia characterized by aggregation of amyloid β (Aβ) and neuronal loss. One of the risk factors for AD is high cholesterol levels, which are known to promote Aβ deposition. Previous studies have shown that royal jelly (RJ), a product of worker bees, has potential neuroprotective effects and can attenuate Aβ toxicity. However, little is known about how RJ regulates Aβ formation and its effects on cholesterol levels and neuronal metabolic activities. Here, we investigated whether RJ can reduce cholesterol levels, regulate Aβ levels and enhance neuronal metabolic activities in an AD rabbit model induced by 2% cholesterol diet plus copper drinking water. Our results suggest that RJ significantly reduced the levels of plasma total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C), and decreased the level of Aβ in rabbit brains. RJ was also shown to markedly ameliorate amyloid deposition in AD rabbits from Aβ immunohistochemistry and thioflavin-T staining. Furthermore, our study suggests that RJ can reduce the expression levels of β-site APP cleaving enzyme-1 (BACE1) and receptor for advanced glycation end products (RAGE), and increase the expression levels of low density lipoprotein receptor-related protein 1 (LRP-1) and insulin degrading enzyme (IDE). In addition, we found that RJ remarkably increased the number of neurons, enhanced antioxidant capacities, inhibited activated-capase-3 protein expression, and enhanced neuronal metabolic activities by increasing N-acetyl aspartate (NAA) and glutamate and by reducing choline and myo-inositol in AD rabbits. Taken together, our data demonstrated that RJ could reduce cholesterol levels, regulate Aβ levels and enhance neuronal metabolic activities in AD rabbits, providing preclinical evidence that RJ treatment has the potential to protect neurons and prevent AD.

Alzheimer’s disease like pathology induced six weeks after aggregated amyloid-beta injection in rats: increased oxidative stress and impaired long-term memory with anxiety-like behavior

Objectives: Amyloid-beta (Aβ) peptide deposition into insoluble plaques is a pathological hallmark of Alzheimer’s disease (AD), but soluble oligomeric Aβ is considered to be more potent and has been hypothesized to directly impair learning and memory. Also, evidences from some clinical studies indicated that Aβ oligomer formation is the major cause for early AD onset. However, the biochemical mechanism involved in the oligomer-induced toxicity is not very well addressed. So, thise present study was undertaken to study the effects of single intracerebroventricular (icv) injection of protofibrillar Aβ 1-42 on the behavioral and biochemical profile in rats.Methods: Rats were divided into two groups (n = 8 per group): (1) sham control group and (2) Aβ 1-42 injected group. A single dose of protofibrillar Aβ 1-42 (5 ul) through icv injection was bilaterally administered into the dorsal hippocampus, while sham control animals were administered with 5 µl of vehicle.Results: The results demonstrated that the protofibrillar Aβ significantly inhibited long-term memory retention and increased anxiety levels as shown by the behavioral studies. The amyloid deposits were present inside the brain even six weeks after injection as confirmed by thioflavin-T staining and the neurodegeneration induced by these deposits was confirmed by Nissl’s staining in hippocampal and cortical regions. The amyloid aggregates induced reactive oxygen species (ROS) production, acetylcholinesterase activity, nitrite levels, lipid peroxidation, and inhibited antioxidant enzyme activity in hippocampus, cortex, and striatum regions of rat brain after six weeks.Discussion: The present study indicated that protofibrillar Aβ 1-42 injection altered long term memory, induced anxiety-like behavior and also developed Alzheimer’s disease like pathology in rats.

Alkaptonuria is a novel human secondary amyloidogenic disease

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Antimicrobial Protegrin-1 Forms Amyloid-Like Fibrils with Rapid Kinetics Suggesting a Functional Link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.

Arginine Metabolising Enzymes as Therapeutic Tools for Alzheimer’s Disease: Peptidyl Arginine Deiminase Catalyses Fibrillogenesis of β-amyloid Peptides

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer's disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of beta-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Abeta(1-40), Abeta(22-35), Abeta(17-28), Abeta(25-35) and Abeta(32-35) but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 micromol min(-1) mg(-1) from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5 degrees C and 68 degrees C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V(max) and K(m) of 1.57 micromol min(-1) ml(-1) and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K(i)) of the amyloid peptides to PAD with values between 1.4 and 4.6 microM. The formation of Abeta fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.

Acetylcholinesterase activity in senile plaques of aged macaques

A modified acetylcholinesterase (AChE)-histochemical technique, which demonstrates axonal morphology to a high degree, was used to examine the neocortices of aged monkeys. This approach disclosed slender linear axonal profiles in young animals. In older monkeys, there was a variety of abnormalities of AChE-containing fibers, including multifocal distentions of individual fibers and aggregations of neuritesized, AChE-rich swellings. Combined with thioflavin-T staining to visualize amyloid, this histochemical technique showed that some of these AChE-containing fibers were present in proximity to deposits of amyloid. This association suggests that abnormal AChE-rich axons participate in the formation of some senile plaques in the neocortices of aged nonhuman primates. While it is probable that many of these AChE-rich fibers are axons of cholinergic neurons residing in the basal forebrain, it is also likely that some of these fibers are derived from noncholinergic neuronal populations known to synthesize AChE. Immunocytochemical strategies can be used to assess the involvement of other systems, including cholinergic, noradrenergic, dopaminergic, somatostatinergic, and serotonergic neurons in the formation of senile plaques in the brains of aged nonhuman primates.

RENAL SEGMENTAL “HYPOPLASIA” (ASK-UPMARK): PATHOLOGIC ASPECTS

Segmental “hypoplasia” of the kidney is a form of severe renal atrophy commonly associated with vesicoureteric reflux (VUR). The scarred segments contain atrophic tubules and hyperplastic blood vessels; glomeruli are often absent or sparse. These lesions have been identified in 21 patients (12F, 9M), 6-27 yr: 13 (62%) hypertensive (HT) and 8 normotensive (NT); 13 with prior urinary tract infection; 18/19 (95%) with radiographic VUR; 8 (38%) with impaired renal function (GFR <40 ml/min/1.73 m2); none with severe proteinuria. Renal atrophy commonly progressed after surgical repair of VUR, even in absence of infection. Lesions were bilateral in 5 HT patients and 2 others had unilateral agenesis; bilateral in only 1 NT patient. HT responded to nephrectomy in all patients. Histologic examination of the scars showed no differences between HT and NT patients in numbers of sclerotic glomeruli or in severity of interstitial fibrosis, arteriolar sclerosis, and inflammatory cell infiltration. Examination of cortical tissue adjacent to scars showed HT patients to have more severe tubular atrophy and vascular change, possibly as the result of HT. Thioflavin-T staining showed the sclerotic glomeruli and hypertrophic arterioles to contain more JGA granules in the scars of HT than of NT patients. Segmental “hypoplasia” appears to be an acquired abnormality, although its association with renal dysplasia in two specimens indicates an occasional origin during intrauterine life.