Pomegranate peel, chokeberry leaves and Ironwort extract as novel natural inhibitors of amylin aggregation and cellular toxicity in pancreatic ß cells.

Impeding or reducing human amylin aggregation and/or its toxicity can be key to preventing pancreatic islet amyloidosis and ß-cell loss in patients with Type 2 Diabetes Mellitus (T2DM). Here, Punica granatum (pomegranate) peel, Sideritis raeseri (ironwort) and Aronia melanocarpa (chokeberry) leaf extracts, were tested for their novel anti-aggregative and antitoxic properties in human amylin (hIAPP) treated rat pancreatic insulinoma (INS) cells. The protein aggregation (Th-T) assay revealed an inhibitory trend of all three plant extracts against amylin aggregates. In agreement with this finding, pomegranate peel and ironwort extracts effectively prevented the transition of hIAPP from disordered, random coil structures into aggregation prone ß-sheet enriched molecular assemblies, revealed by CD spectroscopy. Consistent with their anti-aggregative action, all three extracts prevented, to various degrees, reactive oxygen species (ROS) accumulation, mitochondrial stress, and, ultimately, apoptosis of INS cells. Collectively, the results from this study demonstrate effectiveness of natural products to halt hIAPP aggregation, redox stress, and toxicity, which could be exploited as novel therapeutics against amylin-derived islet amyloidosis and ß-cell stress in T2DM.

Anti-Amyloid Drug Screening Methods Using Bacterial Inclusion Bodies.

Amyloid aggregation is linked to a number of human disorders that range from non-neurological illnesses such as type 2 diabetes to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. The formation of insoluble protein aggregates with amyloid conformation inside bacteria, namely, in bacterial inclusion bodies, offers the possibility to use bacteria as simple models to study amyloid aggregation processes and potential effects of both anti-amyloid drugs and/or pro-aggregative compounds. This chapter describes fast, simple, inexpensive, highly reproducible, and tunable in vitro and in cellulo methods that use bacterial inclusion bodies as preliminary screening tools for anti-amyloid drugs.

Nutrient-sensing amyloid metastasis.

Amyloids are organized suprastructural polypeptide arrangements. The prevalence of amyloid-related processes of pathophysiological relevance has been linked to aging-related degenerative diseases. Besides the role of genetic polymorphisms on the relative risk of amyloid diseases, the contributions of nongenetic ontogenic cluster of factors remain elusive. In recent decades, mounting evidences have been suggesting the role of essential micronutrients, in particular transition metals, in the regulation of amyloidogenic processes, both directly (such as binding to amyloid proteins) or indirectly (such as regulating regulatory partners, processing enzymes, and membrane transporters). The features of transition metals as regulatory cofactors of amyloid proteins and the consequences of metal dyshomeostasis in triggering amyloidogenic processes, as well as the evidences showing amelioration of symptoms by dietary supplementation, suggest an exaptative role of metals in regulating amyloid pathways. The self- and cross-talk replicative nature of these amyloid processes along with their systemic distribution support the concept of their metastatic nature. The role of amyloidosis as nutrient sensors would act as intra- and transgenerational epigenetic metabolic programming factors determining health span and life span, viability, which could participate as an evolutive selective pressure.

Transthyretin: From Structural Stability to Osteoarticular and Cardiovascular Diseases.

Transthyretin (TTR) is a tetrameric protein transporting hormones in the plasma and brain, which has many other activities that have not been fully acknowledged. TTR is a positive indicator of nutrition status and is negatively correlated with inflammation. TTR is a neuroprotective and oxidative-stress-suppressing factor. The TTR structure is destabilized by mutations, oxidative modifications, aging, proteolysis, and metal cations, including Ca2+. Destabilized TTR molecules form amyloid deposits, resulting in senile and familial amyloidopathies. This review links structural stability of TTR with the environmental factors, particularly oxidative stress and Ca2+, and the processes involved in the pathogenesis of TTR-related diseases. The roles of TTR in biomineralization, calcification, and osteoarticular and cardiovascular diseases are broadly discussed. The association of TTR-related diseases and vascular and ligament tissue calcification with TTR levels and TTR structure is presented. It is indicated that unaggregated TTR and TTR amyloid are bound by vicious cycles, and that TTR may have an as yet undetermined role(s) at the crossroads of calcification, blood coagulation, and immune response.

Curcumin: A small molecule with big functionality against amyloid aggregation in neurodegenerative diseases and type 2 diabetes.

Amyloidosis is a concept that implicates disorders and complications that are due to abnormal protein accumulation in different cells and tissues. Protein aggregation-associated diseases are classified according to the type of aggregates and deposition sites, such as neurodegenerative disorders and type 2 diabetes mellitus. Polyphenolic phytochemicals such as curcumin and its derivatives have anti-amyloid effects both in vitro and in animal models; however, the underlying mechanisms are not understood. In this review, we summarized possible mechanisms by which curcumin could interfere with self-assembly processes and reduce amyloid aggregation in amyloidosis. Furthermore, we discuss clinical trials in which curcumin is used as a therapeutic agent for the treatment of diseases linking to protein aggregates.

Subcutaneous amyloidoma models for screening potential anti-fibrillating agents in vivo.

Here, we describe a robust protocol using mouse models to screen potential insulin-stabilizers and insulin moieties. We have generated a mouse model of amyloidoma, found in diabetic patients undergoing insulin therapy. This model can be used to screen potential insulin stabilizers and insulin moieties to prevent amyloidoma formation. This protocol can further be used for the preclinical validation of therapeutically relevant insulin stabilizers and formulations. The protocol highlights all the critical steps for generating amyloidoma in a preclinical model. For complete details on the use and execution of this profile, please refer to Mukherjee et al. (2021).

Dietary Intake of Rosmarinic Acid Increases Serum Inhibitory Activity in Amyloid A Aggregation and Suppresses Deposition in the Organs of Mice.

Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis, although the underlying aggregation mechanism has not been elucidated. Since SAA aggregation is a key step in this pathogenesis, inhibitors are useful to prevent and treat AA amyloidosis, serving as tools to investigate the pathogenic mechanism. In this study, we showed that rosmarinic acid (RA), which is a well-known inhibitor of the aggregation of amyloid ß (Aß), displayed inhibitory activity against SAA aggregation in vitro using a microliter-scale high-throughput screening (MSHTS) system with quantum-dot nanoprobes. Therefore, we evaluated the amyloid aggregation inhibitory activity of blood and the deposition of SAA in organs by feeding mice with Melissa officinalis extract (ME) containing RA as an active substance. Interestingly, the inhibitory activity of ME-fed mice sera for SAA and Aß aggregation, measured with the MSHTS system, was higher than that of the control group. The amount of amyloid deposition in the organs of ME-fed mice was lower than that in the control group, suggesting that the SAA aggregation inhibitory activity of serum is associated with SAA deposition. These results suggest that dietary intake of RA-containing ME enhanced amyloid aggregation inhibitory activity of blood and suppressed SAA deposition in organs. This study also demonstrated that the MSHTS system could be applied to in vitro screening and to monitor comprehensive activity of metabolized foods adsorbed by blood.

The Protective Effects of Acer okamotoanum and Isoquercitrin on Obesity and Amyloidosis in a Mouse Model.

Obesity increases risk of Alzheimer's Disease (AD). A high fat diet (HFD) can lead to amyloidosis and amyloid beta (Aß) accumulation, which are hallmarks of AD. In this study, protective effects of the ethyl acetate fraction of Acer okamotoanum (EAO) and isoquercitrin were evaluated on obesity and amyloidosis in the HFD- and Aß-induced mouse model. To induce obesity and AD by HFD and Aß, mice were provided with HFD for 10 weeks and were intracerebroventricularly injected with Aß25-35. For four weeks, 100 and 10 mg/kg/day of EAO and isoquercitrin, respectively, were administered orally. Administration of EAO and isoquercitrin significantly decreased body weight in HFD and Aß-injected mice. Additionally, EAO- and isoquercitrin-administered groups attenuated abnormal adipokines release via a decrease in leptin and an increase in adiponectin levels compared with the control group. Furthermore, HFD and Aß-injected mice had damaged liver tissues, but EAO- and isoquercitrin-administered groups attenuated liver damage. Moreover, administration of EAO and isoquercitrin groups down-regulated amyloidosis-related proteins in the brain such as ß-secretase, presenilin (PS)-1 and PS-2 compared with HFD and Aß-injected mice. This study indicated that EAO and isoquercitrin attenuated HFD and Aß-induced obesity and amyloidosis, suggesting that they could be effective in preventing and treating both obesity and AD.

Arginase 1 Insufficiency Precipitates Amyloid-ß Deposition and Hastens Behavioral Impairment in a Mouse Model of Amyloidosis.

Alzheimer's disease (AD) includes several hallmarks comprised of amyloid-ß (Aß) deposition, tau neuropathology, inflammation, and memory impairment. Brain metabolism becomes uncoupled due to aging and other AD risk factors, which ultimately lead to impaired protein clearance and aggregation. Increasing evidence indicates a role of arginine metabolism in AD, where arginases are key enzymes in neurons and glia capable of depleting arginine and producing ornithine and polyamines. However, currently, it remains unknown if the reduction of arginase 1 (Arg1) in myeloid cell impacts amyloidosis. Herein, we produced haploinsufficiency of Arg1 by the hemizygous deletion in myeloid cells using Arg1fl/fl and LysMcreTg/+ mice crossed with APP Tg2576 mice. Our data indicated that Arg1 haploinsufficiency promoted Aß deposition, exacerbated some behavioral impairment, and decreased components of Ragulator-Rag complex involved in mechanistic target of rapamycin complex 1 (mTORC1) signaling and autophagy. Additionally, Arg1 repression and arginine supplementation both impaired microglial phagocytosis in vitro. These data suggest that proper function of Arg1 and arginine metabolism in myeloid cells remains essential to restrict amyloidosis.

Characterization of the unique In Vitro effects of unsaturated fatty acids on the formation of amyloid ß fibrils.

Accumulation of amyloid ß (Aß) peptides, the major component of amyloid fibrils in senile plaques, is one of the main causes of Alzheimer's disease. Docosahexaenoic acid (DHA) is a fatty acid abundant in the brain, and is reported to have protective effects against Alzheimer's disease, although the mechanistic effects of DHA against Alzheimer's pathophysiology remain unclear. Because dietary supplementation of DHA in Aß precursor protein transgenic mice ameliorates Aß pathology and behavioral deficits, we hypothesize that DHA may affect the fibrillization and deposition of Aß. Here we studied the effect of different types of fatty acids on Aß fibril formation by in vitro Aß fibrillization assay. Formation of amyloid fibrils consists of two steps, i.e., the initial nucleation phase and the following elongation phase. We found that unsaturated fatty acids, especially DHA, accelerated the formation of Aß fibrils with a unique short and curved morphology in its nucleation phase, which did not elongate further into the long and straight, mature Aß fibrils. Addition of DHA afterwards did not modify the morphology of the mature Aß(1-40) fibrils. The short and curved Aß fibrils formed in the presence of DHA did not facilitate the elongation phase of Aß fibril formation, suggesting that DHA promotes the formation of "off-pathway" conformers of Aß. Our study unravels a possible mechanism of how DHA acts protectively against the pathophysiology of Alzheimer's disease.

Structural Stabilization of Human Transthyretin by Centella asiatica (L.) Urban Extract: Implications for TTR Amyloidosis.

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic ß-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with ß-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic ß-cells. Furthermore, AZD supplementation in pancreatic ß-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

Use of Curcumin, a Natural Polyphenol for Targeting Molecular Pathways in Treating Age-Related Neurodegenerative Diseases.

Progressive accumulation of misfolded amyloid proteins in intracellular and extracellular spaces is one of the principal reasons for synaptic damage and impairment of neuronal communication in several neurodegenerative diseases. Effective treatments for these diseases are still lacking but remain the focus of much active investigation. Despite testing several synthesized compounds, small molecules, and drugs over the past few decades, very few of them can inhibit aggregation of amyloid proteins and lessen their neurotoxic effects. Recently, the natural polyphenol curcumin (Cur) has been shown to be a promising anti-amyloid, anti-inflammatory and neuroprotective agent for several neurodegenerative diseases. Because of its pleotropic actions on the central nervous system, including preferential binding to amyloid proteins, Cur is being touted as a promising treatment for age-related brain diseases. Here, we focus on molecular targeting of Cur to reduce amyloid burden, rescue neuronal damage, and restore normal cognitive and sensory motor functions in different animal models of neurodegenerative diseases. We specifically highlight Cur as a potential treatment for Alzheimer's, Parkinson's, Huntington's, and prion diseases. In addition, we discuss the major issues and limitations of using Cur for treating these diseases, along with ways of circumventing those shortcomings. Finally, we provide specific recommendations for optimal dosing with Cur for treating neurological diseases.

[Protein carbamylation: what it is and why it concerns nephrologists].

Spontaneous urea dissociation in water solution is a prominent source of protein carbamylation in our body. Protein carbamylation is a well-known phenomenon since early seventies. Some years ago, much interest in the diagnostic power of carbamylated protein arouse. Recently the target of the researches focused on its potential cardiovascular pathogenicity. Some authors claimed that this could be a reason for higher cardiovascular mortality in uremic patients. Nutritional therapy, amino acids supplementation and intensive dialysis regimen are some of the therapeutic tools tested to lower the carbamylation burst in this population.

The hypothalamus as the primary brain region of metabolic abnormalities in APP/PS1 transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an amyloid-related neurodegenerative disorder and is also considered to be a metabolic disease. Thus, investigation of metabolic mechanisms of amyloid pathology progression is of substantial importance for the diagnosis, prevention and treatment of AD. In the present study, cognitive function and brain metabolism were explored in the transgenic APP/PS1 mouse model of amyloid pathology at different ages. Using an NMR-based metabolomic approach, we examined metabolic changes in six different brain regions of wild-type and APP/PS1 mice at 1, 5 and 10months of age. Learning and memory performance in mice was evaluated using the Morris water maze test. Furthermore, a generalized linear mixed model was employed to analyze the interaction effect between the mouse-type and brain region (or age) on metabolic alterations. Brain region-specific changes in energy metabolism occurred prior to a very early-stage of amyloid pathology (1month of age) in APP/PS1 mice. A hypermetabolic state was identified in the brains of APP/PS1 mice at 5months of age, and the hypothalamus was identified as the main brain region that underwent significant metabolic alterations. The cognitive function of APP/PS1 mice was impaired at 10months of age; moreover, the hypermetabolic state identified in various brain regions at 5months of age was also significantly decreased. In conclusion, our results suggest that a hypothalamic metabolism abnormality may comprise a potential indicator for the early-diagnosis and monitoring of amyloid pathology progression.

Applications of the real-time quaking-induced conversion assay in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

INTRODUCTION: The development of in vitro protein misfolding amplification assays for the detection and analysis of abnormally folded proteins, such as proteinase K resistant prion protein (PrPres) was a major innovation in the prion field. In prion diseases, these types of assays imitate the pathological conversion of the cellular PrP (PrPC) into a proteinase resistant associated conformer or amyloid, called PrPres. Areas covered: The most prominent protein misfolding amplification assays are the protein misfolding cyclic amplification (PMCA), which is based on sonication and the real-time quaking-induced conversion (RT-QuIC) technique based on shaking. The more recently established RT-QuIC is fully automatic and enables the monitoring of misfolded protein aggregates in real-time by using a fluorescent dye. Expert commentary: RT-QuIC is a very robust and highly reproducible test system which is applicable in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

Natural product-based amyloid inhibitors.

Many chronic human diseases, including multiple neurodegenerative diseases, are associated with deleterious protein aggregates, also called protein amyloids. One common therapeutic strategy is to develop protein aggregation inhibitors that can slow down, prevent, or remodel toxic amyloids. Natural products are a major class of amyloid inhibitors, and several dozens of natural product-based amyloid inhibitors have been identified and characterized in recent years. These plant- or microorganism-extracted compounds have shown significant therapeutic potential from in vitro studies as well as in vivo animal tests. Despite the technical challenges of intrinsic disordered or partially unfolded amyloid proteins that are less amenable to characterizations by structural biology, a significant amount of research has been performed, yielding biochemical and pharmacological insights into how inhibitors function. This review aims to summarize recent progress in natural product-based amyloid inhibitors and to analyze their mechanisms of inhibition in vitro. Major classes of natural product inhibitors and how they were identified are described. Our analyses comprehensively address the molecular interactions between the inhibitors and relevant amyloidogenic proteins. These interactions are delineated at molecular and atomic levels, which include covalent, non-covalent, and metal-mediated mechanisms. In vivo animal studies and clinical trials have been summarized as an extension. To enhance natural product bioavailability in vivo, emerging work using nanocarriers for delivery has also been described. Finally, issues and challenges as well as future development of such inhibitors are envisioned.

Systemic Amyloidosis Model on Young Mice.

Subcutaneous daily injection (with neglect of aseptics) of 0.5 ml solution of soybean cream substitute (10% volume in distilled water) during 30 days caused systemic amyloidosis in 30-day-old mice. All the known methods for induction of systemic amyloidosis are based on the use of old animals, as senile tissue bradytrophy allows effective simulation of amyloidosis.

Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo.

Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.