Amyloid Degradation Research Articles

Degradation of pathogenic amyloids induced by matrix metalloproteinase-9

Over the past decade, the greatest promise for treating severe and currently incurable systemic and neurodegenerative diseases has turned to agents capable of effectively degrading pathological amyloid deposits without causing side effects. Specifically, amyloid destruction observed in immunotherapy is hypothesized to occur through activation of proteolytic enzymes. This study examines poorly understood effects of an immune enzyme, extracellular matrix metalloproteinase-9 (MMP9), on amyloids associated with Alzheimer's and Parkinson's diseases, lysozyme, insulin, and dialysis-related amyloidoses. The study establishes the universality of MMP9's effect on various amyloids, with its efficacy largely depending on the fibrillar cluster size. Irreversible amyloid degradation by MMP9 is attributed to the destruction of intramolecular interactions rather than intermolecular hydrogen bonds in the fibril backbone. This process results in the loss of ordered fiber structure without reducing aggregate size or increasing cytotoxicity. Thus, MMP9 can mitigate side effects of anti-amyloid therapy associated with the formation of low-molecular-weight degradation products that may accelerate fibrillogenesis and amyloid propagation between tissues and organs. MMP9 shows promise as a component of safe anti-amyloid drugs by enhancing the accessibility of binding sites through “loosening” amyloid clusters, which facilitates subsequent fragmentation and monomerization by other enzymes.

Exploring the amyloid degradation potential of nanoformulated carrageenan-bridging in vitro and in vivo perspectives

Amyloids, with their β-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17–20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

The Role of Mesenchymal Stem Cell Exosomes in the Onset and Progression of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease with progressive loss of memory and cognitive function. Because its pathogenesis has not been fully elucidated, there is still a lack of understanding of its pathogenesis and effective treatment. Many studies have shown that Mesenchymal stem cells Exosomes (MSCs-Exo) can promote anti-inflammatory, regulate immune function, enhance amyloid (Aβ) degradation, and promote axon growth of nerve cells. Exosomes can effectively cross the blood-brain barrier, and have better biocompatibility and biosafety than nanomaterials, and more diverse ways to carry drugs. Adipose stem cell exosomes can not only treat AD by their own characteristics, but also regulate AD and other neurological diseases as A drug carrier that can efficiently and freely cross the blood-brain barrier: they can effectively reduce Aβ42 plaques and improve the neuronal microenvironment through microglia. In AD model mice, excessive activation of microglia demonstrated the formation of an inflammatory microenvironment in the brain. The inflammatory microenvironment erodes healthy neurons to a certain extent, forcing them to overactivate inflammatory signaling pathways so that they can't function normally, Adipose stem cell exosomes can effectively clear the expression of inflammatory factors in hypoxia-induced AD neuronal model and promote synaptic repair through microRNA/ phosphatase and tensin homolog (miR-223/PTEN) and phosphatidylinositol kinase (PI3K/Akt) pathways. It can promote the polarization of microglia towards anti-inflammatory phenotype and promote neuronal repair. Adipose stem cell exosomes can also be used to improve memory and cognitive dysfunction by alleviating Tau phosphating or delaying hippocampus volume decay, targeting the core pathological mechanism of AD to achieve therapeutic effects. This article mainly introduces the role of MSC exosomes in the pathogenesis and development of AD.

In Vitro Astroglial Dysfunction Induced by Neurotoxins: Mimicking Astrocytic Metabolic Alterations of Alzheimer's Disease.

Astrocytes play fundamental roles in the maintenance of brain homeostasis. The dysfunction of these cells is widely associated with brain disorders, which are often characterized by variations in the astrocyte protein markers GFAP and S100B, in addition to alterations in some of its metabolic functions. To understand the role of astrocytes in neurodegeneration mechanisms, we induced some of these metabolic alterations, such as energy metabolism, using methylglyoxal (MG) or fluorocitrate (FC); and neuroinflammation, using lipopolysaccharide (LPS) and streptozotocin (STZ), which is used for inducing Alzheimer's disease (AD) in animal models. We showed that MG, LPS, STZ and FC similarly caused astrocyte dysfunction by increasing GFAP and reducing S100B secretion. In the context of AD, STZ caused an amyloid metabolism impairment verified by increases in Aβ1-40 peptide content and decreases in the amyloid degradation enzymes, IDE and NEP. Our data contribute to the understanding of the role of astrocytes in brain injury mechanisms and suggest that STZ is suitable for use in vitro models for studying the role of astrocytes in AD.

Towards the Integrative Theory of Alzheimer's Disease: Linking Molecular Mechanisms of Neurotoxicity, Beta-amyloid Biomarkers, and the Diagnosis.

A major gap in amyloid-centric theories of Alzheimer's disease (AD) is that even though amyloid fibrils per se are not toxic in vitro, the diagnosis of AD clearly correlates with the density of beta-amyloid (Aβ) deposits. Based on our proposed amyloid degradation toxicity hypothesis, we developed a mathematical model explaining this discrepancy. It suggests that cytotoxicity depends on the cellular uptake of soluble Aβ rather than on the presence of amyloid aggregates. The dynamics of soluble beta-amyloid in the cerebrospinal fluid (CSF) and the density of Aβ deposits is described using a system of differential equations. In the model, cytotoxic damage is proportional to the cellular uptake of Aβ, while the probability of an AD diagnosis is defined by the Aβ cytotoxicity accumulated over the duration of the disease. After uptake, Aβ is concentrated intralysosomally, promoting the formation of fibrillation seeds inside cells. These seeds cannot be digested and are either accumulated intracellularly or exocytosed. Aβ starts aggregating on the extracellular seeds and, therefore, decreases in concentration in the interstitial fluid. The dependence of both Aβ toxicity and aggregation on the same process-cellular uptake of Aβ-explains the correlation between AD diagnosis and the density of amyloid aggregates in the brain. We tested the model using clinical data obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI), which included records of beta-amyloid concentration in the cerebrospinal fluid (CSF-Aβ42) and the density of beta-amyloid deposits measured using positron emission tomography (PET). The model predicts the probability of AD diagnosis as a function of CSF-Aβ42 and PET and fits the experimental data at the 95% confidence level. Our study shows that existing clinical data allows for the inference of kinetic parameters describing beta-amyloid turnover and disease progression. Each combination of CSF-Aβ42 and PET values can be used to calculate the individual's cellular uptake rate, the effective disease duration, and the accumulated toxicity. We show that natural limitations on these parameters explain the characteristic distribution of the clinical dataset for these two biomarkers in the population. The resulting mathematical model interprets the positive correlation between the density of Aβ deposits and the probability of an AD diagnosis without assuming any cytotoxicity of the aggregated beta-amyloid. To the best of our knowledge, this model is the first to mechanistically explain the negative correlation between the concentration of Aβ42 in the CSF and the probability of an AD diagnosis. Finally, based on the amyloid degradation toxicity hypothesis and the insights provided by mathematical modeling, we propose new pathophysiology-relevant biomarkers to diagnose and predict AD.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aβ-peptide (Aβ42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

Metal Complexes as Promising Matrix Metalloproteinases Regulators.

Nowadays, cancers and dementia, such as Alzheimer's disease, are the most fatal causes of death. Many studies tried to understand the pathogenesis of those diseases clearly and develop a promising way to treat the diseases. Matrix metalloproteinases (MMPs) have been reported to be involved in the pathology of cancers and AD through tumor cell movement and amyloid degradation. Therefore, control of the levels and actions of MMPs, especially MMP-2 and MMP-9, is necessary to care for and/or cure cancer and AD. Various molecules have been examined for their potential application as regulators of MMPs expression and activity. Among the molecules, multiple metal complexes have shown advantages, including simple synthesis, less toxicity and specificity toward MMPs in cancer cells or in the brain. In this review, we summarize the recent studies and knowledge of metal complexes (e.g., Pt-, Ru-, Au-, Fe-, Cu-, Ni-, Zn-, and Sn-complexes) targeting MMPs and their potentials for treating and/or caring the most fatal human diseases, cancers and AD.

Catalysis medicine: Participating in the chemical networks of living organisms through catalysts

We are investigating catalytic approaches to participating in the chemical networks of living organisms. Catalysts that enable us to do so activate stable molecules with high selectivity under physiological conditions and will form the basis of a new area of medicine, catalysis medicine. Additionally, our research will contribute to progress in both physical and life sciences based on catalysis. In this Perspective, we provide a general overview of the concept of catalysis medicine, with examples of our recent achievements in chemical catalyst-promoted synthetic epigenetics in living cells and amyloid degradation in Alzheimer's disease model mice. This Perspective postulates the potential for catalysis research to act as a foundation from which to tackle important interdisciplinary problems.

Macrophages in the reticuloendothelial system inhibit early induction stages of mouse apolipoprotein A-II amyloidosis

Amyloidosis refers to a group of degenerative diseases that are characterized by the deposition of misfolded protein fibrils in various organs. Deposited amyloid may be removed by a phagocyte-dependent innate immune system; however, the precise mechanisms during disease progression remain unclear. We herein investigated the properties of macrophages that contribute to amyloid degradation and disease progression using inducible apolipoprotein A-II amyloidosis model mice. Intravenously injected AApoAII amyloid was efficiently engulfed by reticuloendothelial macrophages in the liver and spleen and disappeared by 24 h. While cultured murine macrophages degraded AApoAII via the endosomal-lysosomal pathway, AApoAII fibrils reduced cell viability and phagocytic capacity. Furthermore, the depletion of reticuloendothelial macrophages before the induction of AApoAII markedly increased hepatic and splenic AApoAII deposition. These results highlight the physiological role of reticuloendothelial macrophages in the early stages of pathogenesis and suggest the maintenance of phagocytic integrity as a therapeutic strategy to inhibit disease progression.

Bridging the gap between cellular amyloid metabolism and Alzheimer’s disease clinical data

AbstractBackgroundAlzheimer’s disease (AD) diagnosis strongly correlates with the density of amyloid deposits. Yet, amyloid fibrils per se are not toxic, which represents a major gap in amyloid‐based theories of AD. Based on the proposed previously amyloid degradation toxicity hypothesis, we present the mathematical model of amyloid turnover that bridges this gap (Fig.1).MethodsThe model suggests that the probability of AD diagnosis is defined by amyloid cytotoxicity accumulated over sickness duration. The cytotoxicity depends on the cellular uptake of soluble beta‐amyloid rather than on amyloid aggregates. Also, after uptake, beta‐amyloid is concentrated intralysosomally and forms fibrillation seeds. They cannot be digested and after exocytosis grow by aggregating more beta‐amyloid. The equations describing the model are solved analytically using two major AD biomarkers as independent variables: beta‐amyloid concentration in the cerebrospinal fluid (CSF‐Aβ42) and amyloid deposits density measured by positron emission tomography (PET). Parameters of the model are inferred using maximum likelihood estimation method from the clinical dataset obtained by ADNI.ResultsThe model describes the probability of AD diagnosis as a function of CSF‐Aβ42 and PET and confirms major previously formulated clinical observations (Fig.2). (1) The probability of AD increases with PET, i.e., the density of amyloid deposits; (2) For participants with the same PET, the probability of AD is higher at lower CSF‐Aβ42 levels. For each pair of values (CSF‐Aβ42; PET), the model allows calculating cellular uptake, sickness duration, and accumulated toxicity. Natural limitations on these parameters explain the distribution of CSF‐Aβ42 and PET (Fig.3). Specifically, these values are bound by (1) minimal and maximal cellular uptake; (2) high accumulated toxicity; 3) disease duration.ConclusionsExisting clinical data allow the inference of kinetic parameters describing beta‐amyloid turnover. The resulting mathematical model predicts positive correlation between the density of beta‐amyloid aggregates and the probability of AD diagnosis in absence of cytotoxicity of amyloid deposits. To the best of our knowledge, this model is the first to mechanistically explain negative correlation between the probability of AD diagnosis and CSF concentration of soluble beta‐amyloid. Finally, natural limitations on model parameters explain the overall distribution of measured CSF‐Aβ42 and PET values.

Exercise Benefits on Alzheimer’s Disease

Alzheimer’s disease (AD) is a major threat to the health of the elderly, and there are few drugs that can completely cure AD. Here we find that longterm exercise improves cognitive impairment and motor coordination in AD mice, maintains lysosomal Golgi morphology, promotes lysosomal maturation to enhance lysosomal function, stimulates enhanced effects of TFEB and AMPK mediated acetyl-CoA synthetase2 (ACSS2) for lysosomal biogenesis thereby increasing amyloid degradation and reducing its accumulation in the hippocampus and cortex.

Cardiac Amyloidosis Treatment.

Cardiac amyloidosis (CA) is a restrictive cardiomyopathy with a traditionally poor prognosis. Until recently, CA treatment options were limited and consisted predominantly of managing symptoms and disease-related complications. However, the last decade has seen significant advances in disease-modifying therapies, increased awareness of CA, and improved diagnostic methods resulting in earlier diagnoses. In this review, we provide an overview of current and experimental treatments for the predominant types of CA: transthyretin cardiac amyloidosis (ATTR-CA) and immunoglobulin light chain (AL)-mediated CA (AL-CA).The mainstay of AL-CA treatment is proteasome inhibitor-based chemotherapy with daratumumab and, when feasible, autologous stem cell transplantation. For ATTR-CA, the stabilizer tafamidis is the only US Food and Drug Administration (FDA)-approved treatment. However, promising novel therapies on the horizon target various points in the ATTR-CA amyloidogenic cascade. These include transthyretin gene (TTR) silencing agents to prevent TTR formation, TTR tetramer stabilization and inhibition of oligomer aggregation to prevent fibril formation, anti-TTR fiber antibodies, and amyloid degradation. For end-stage CA, advanced interventions may need to be considered, including heart, heart-kidney, and, for hereditary ATTR-CA, heart-liver transplantation. Despite the evolution of treatment options, CA management remains complex due to patient frailty and therapeutic side effects or intolerance with advanced cardiac disease. This is particularly relevant for those with AL-CA, when active teamwork between the hematologist-oncologist and the cardiologist is critical for treatment success. Often, referral to an expert center is necessary for timely diagnosis, initiation of treatment, and participation in clinical trials.

Lysosomal permeabilization as a mechanism of amyloid degradation toxicity in Alzheimer's disease.

The amyloid channel theory readily explains primary molecular damage induced by beta-amyloid (Aβ) but cannot interpret multiple major phenomena associated with Alzheimer's Disease such as autophagy failure and decreased metabolism. To explain them, the amyloid degradation toxicity hypothesis suggests that the cytotoxicity is initiated by the channel formation in lysosomal membranes by amyloid fragments (such as Aβ25-35 ) produced by the digestion of endocytosed Aβ. One amyloid channel is sufficient to neutralize lysosomal content and inactivate proteases; therefore, undigested amyloid accumulates, and autophagy stalls. Inadequate mitophagy results in an increased generation of reactive oxygen species and decreased energy production. Huge electrical conductance of amyloid channels prompted us to estimate the maximal size of the macromolecules which can leak from permeabilized lysosomes through amyloid channels. To estimate the channels' ability to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in the non-conductive membranes passing spheres with a density of average globular proteins. The histograms of single channel conductance at various pH were constructed from the previously published experimental dataset. An approximation with a power function was used to extrapolate the probabilities of channels with high molecular weight cut-offs (MWCO). A variability of channel sizes suggests that channel-forming barrel-shaped aggregates can have various number of monomers. Extrapolation of the channel size distribution predicts that a significant number of channels has MWCO exceeding 10 kDa, while acidic environment disproportionally promotes their formation. This range of MWCO is sufficient for the leakage of cathepsins (20-30 kDa) implicated in the induction of necrosis and/or apoptosis. Channels with MWCO above 1000 Da can release into the cytoplasm the amyloid fragments which can form membrane channels in the plasma membrane by entering from the internal leaflet. The time needed for endocytosis and proteolytic digestion explains why cellular responses to Aβ exposure are taking significant time (at least several minutes). While dissipation of the proton gradient by small amyloid channel readily explains lysosomal failure, relatively rare events of lysosomal permeabilization to large macromolecules by amyloid channels can be an alternative mechanism of cellular death induced by the exposure to Aβ.

Membrane channel hypothesis of lysosomal permeabilization by beta-amyloid

Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-β (Aβ) peptides. Recently, we proposed the amyloid degradation toxicity hypothesis, which explains multiple major observations associated with AD including autophagy failure and a decreased metabolism. According to the hypothesis, the key event in the cellular toxicity of amyloid is the formation of non-selective membrane channels in lysosomal membranes by amyloid fragments that are produced by the digestion of Aβ previously absorbed by endocytosis. Electrophysiological data suggest that amyloid-formed channels have different sizes, which can be explained by the fact that channel creating barrel-shaped amyloid aggregates can consist of different number of monomers.To estimate the ability of channels to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in non-conductive membranes that pass spheres with a density of average globular proteins. As a basis, we used the conductance distribution taken from the previously published experimental dataset, in which single channels with electrical conductance of up to one nanosiemens were registered. Our calculations show that channels with such a giant conductance can allow for passing macromolecules such as large as lysosomal cathepsins implicated in the activation of apoptosis. The formation of giant channels is disproportionally promoted in an acidic environment. Also, amyloid fragments leaking from permeabilized lysosomes can reach the internal leaflet of the plasma membrane and permeabilize it.We conclude that while dissipation of the proton gradient by any (even smallest) amyloid channels readily explains lysosomal failure, the relatively rare events of lysosomal permeabilization to large macromolecules can be an additional mechanism of cellular death induced by exposure to Aβ.

Identifying the Main Functional Pathways Associated with Cognitive Resilience to Alzheimer's Disease.

Understanding the mechanisms involved in cognitive resilience in Alzheimer’s disease (AD) represents a promising strategy to identify novel treatments for dementia in AD. Previous findings from our group revealed that the study of aged-Tg2576 cognitive resilient individuals is a suitable tool for this purpose. In the present study, we performed a transcriptomic analysis using the prefrontal cortex of demented and resilient Tg2576 transgenic AD mice. We have been able to hypothesize that pathways involved in inflammation, amyloid degradation, memory function, and neurotransmission may be playing a role on cognitive resilience in AD. Intriguingly, the results obtained in this study are suggestive of a reduction of the influx of peripheral immune cells into the brain on cognitive resilient subjects. Indeed, CD4 mRNA expression is significantly reduced on Tg2576 mice with cognitive resilience. For further validation of this result, we analyzed CD4 expression in human AD samples, including temporal cortex and peripheral blood mononuclear cells (PBMC). Interestingly, we have found a negative correlation between CD4 mRNA levels in the periphery and the score in the Mini-Mental State Exam of AD patients. These findings highlight the importance of understanding the role of the immune system on the development of neurodegenerative diseases and points out to the infiltration of CD4+ cells in the brain as a key player of cognitive dysfunction in AD.

Effect of metallic nanoparticles on amyloid fibrils and their influence to neural cell toxicity

The modification of amyloid fibrils cytotoxicity through exogenous nanomaterials is crucial to understand the processes controlling the role of protein aggregation in the related diseases. The influence of nanoparticles on amyloid stability yields great interest due to the small size and high surface area-to-volume ratio of nanoparticles. Various physico-chemical parameters play a role in the interaction of proteins and nanoparticles in solution, thus influencing the disaggregation of preformed fibrils. We have examined the influence of two kinds of metallic nanoparticles on lysozyme amyloid fibrils using a multi-technique approach and focalized their impact on cytotoxicity on human neuroblastoma cells (SH-SY5Y). In particular, fluorescence, infrared and circular dichroism spectroscopies, optical and atomic force microscopy experiments have been carried out; the results are analyzed to rationalize the effects of these complexes on neural cell viability. It is remarkable, that the fibrils in the presence of AuNPs, unlike fibrils alone or with AgNPs, do not generate a significant cytotoxic effect even at high concentration and an amyloid degradation effect is visible.

Interaction of ZnO Nanostructures with Proteins: In Vitro Fibrillation/Antifibrillation Studies and in Silico Molecular Docking Simulations.

Protein amyloidosis is related to many neurological disorders. Nanoparticles (NPs) due to their small size can regulate both the polypeptide monomers/oligomers assembly into amyloid fibrils/plaques and the disintegration of the existent plaques. Herein, we have synthesized ZnO nanoflowers and polyol-coated ZnO NPs of relatively small size (40 nm) with cylindrical shape, through solvothermal and microwave-assisted routes, respectively. The effect of the different morphology of nanostructures on the fibrillation/antifibrillation process was monitored in bovine serum albumin (BSA) and human insulin (HI) by fluorescence Thioflavin T (ThT) measurements. Although both nanomaterials affected the amyloid formation mechanism as well as their disaggregation, ZnO nanoflowers with their sharp edges exhibited the greatest amyloid degradation rate in both model proteins (73% and 35%, respectively) and inhibited the most the insulin fibril growth, while restrained also the fibrillation process in the case of albumin solution. In silico molecular docking simulations on the crystal structure of BSA and HI were performed to analyze further the observed in vitro activity of ZnO nanostructures. The binding energy of ZnO NPs was found lower for BSA (-5.44), highlighting their ability to act as catalysts in the fibrillation process of albumin monomers.

The amyloid-\u03b2 degradation intermediate A\u03b234 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer\u2019s disease

An impairment of amyloid β-peptide (Aβ) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer’s disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aβ40 and Aβ42 results in the formation of a common Aβ34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aβ34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aβ34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aβ34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aβ34 immunoreactivity was largely lost. Aβ34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aβ40, but not with Aβ42 levels. Moreover, a significantly decreased Aβ34/Aβ40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aβ40 to Aβ34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aβ34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aβ34 levels upon treatment with recombinant Aβ40 peptides while Aβ34 production was impaired when Aβ40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aβ34 is generated by a novel BACE1-mediated Aβ clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.

Interplay between Amyloid Fibrillation Delay and Degradation by Magnetic Zinc-Doped Ferrite Nanoparticles.

Amyloidosis, the aggregation of naturally soluble proteins into fibrils, is the main pathological hallmark of central nervous system (CNS) disorders, and new therapeutic approaches can be introduced through nanotechnology. Herein, magnetic nanoparticles (MNPs) are proposed to combat amyloidosis and act as CNS theranostic (therapy and diagnosis) candidates through magnetomechanical forces that can be induced under a low-frequency magnetic field. In that vein, a modified one-step microwave-assisted polyol process has been employed to synthesize hybrid organic/inorganic zinc ferrite (ZnxFe3-xO4) MNPs with different levels of zinc doping (0.30 < x < 0.6) derived from the utilized polyol. The lowest doped (x = 0.30) MNPs exhibited high magnetization (127 emu/g), high T2 imaging ability (r2 = 432 mM-1 s-1), and relatively small hydrodynamic size (180 nm), decisive characteristics to further evaluate their CNS theranostic potential. Their effect on the fibrillation/degradation was monitored in two model proteins, insulin and albumin, in the presence/absence of variant external magnetic fields (static, rotating, or alternating) via Thioflavin T (ThT) fluorescence assay and optical fluorescence microscopy. The MNPs were injected either in oligomer solution where significant fibrillation delay was observed, boosted by zinc ionic leaching of MNPs, or in already formed amyloid plaques where up to 86% amyloid degradation was recorded in the presence of magnetic fields, unveiling magnetomechanical antifibrillation properties. The alternating magnetic field (4 Hz) allows the bouncing of the MNPs into the amyloid net driven by the magnetic forces, and thus is featured as the preferred "dancing mode", which strengthens the degrading efficacy of MNPs.

ZnO Nanoflower petals mediated amyloid degradation - An in vitro electrokinetic potential approach

An electrokinetic potential (ζ-potential) based approach was introduced to address the amyloid degradation on ZnO-nanoflower platform. The hallmark of neurodegenerative disorders like Alzheimer's disease, Parkinson's disease (PD), Creutzfeldt-Jakob Disease (CJD), Prion- associated diseases, type-II diabetes, etc. is the deposition of misfolded protein aggregates predominantly β-sheeted in structure and fibrillar morphology, known as amyloids, in the brain and different parts of the body. Agents that can degrade these amyloids can be potential candidate for the therapy of amyloidosis. Ultrasmall nanoparticles are gaining interest due to their ability to cross blood brain barrier (BBB) which is favorable for the treatment of neurodegenerative disorders. Considering the influence of Zn2+ in the formation of Aβ aggregates instead of fibrillation, the present study was designed based on the ZnO nanoparticles (ZnO-NP) and ZnO nanoflowers (ZnO-NF) to compare the anti amyloid ability using a model huminsulin amyloid. Fluorescence study, atomic force microscopy (AFM), IR spectroscopy (FTIR) and reduction of fibril size using dynamic light scattering showed that ZnO-NF can degrade amyloids with a higher capacity than their nanoparticle counterpart. Significant reduction in magnitude of ζ-potential in ZnO-NF treated huminsulin amyloid supported the notion to come to the consensus and became the new indicator for anti-amyloidosis. The cell viability assay of ZnO-NP and ZnO-NF at a higher dose than that used for amyloid degradation using PC12 and HaCaT cell lines showed their biocompatibility in a safe manner. Thus, it can be suggested that ZnO-NF would be a better candidate for amyloid degradation compared to ZnO-NPs due to higher surface to volume ratio of the petals.