Amyloid-associated Disease Research Articles

Reversing pathology in a preclinical model of Alzheimer's disease by hacking cerebrovascular neoangiogenesis with advanced cancer therapeutics.

BackgroundCognitive decline leading to dementia, accompanied by the accumulation of amyloid-beta (Aβ) in neuritic plaques together with the appearance of neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein (tau), are previously noted hallmarks of Alzheimer's disease (AD). We previously discovered hypervascularity in brain specimens from AD patients and consistent with this observation, we demonstrated that overexpression of Aβ drives cerebrovascular neoangiogenesis leading to hypervascularity and coincident tight-junction disruption and blood-brain barrier (BBB) leakiness in animal models of AD. We subsequently demonstrated that amyloid plaque burden and cerebrovascular pathogenesis subside when pro-angiogenic Aβ levels are reduced. Based on these data, we propose a paradigm of AD etiology where, as a compensatory response to impaired cerebral blood flow (CBF), Aβ triggers pathogenic cerebrovascular neoangiogenesis that underlies the conventional hallmarks of AD. Consequently, here we present evidence that repurposing anti-cancer drugs to modulate cerebrovascular neoangiogenesis, rather than directly targeting the amyloid cascade, may provide an effective treatment for AD and related vascular diseases of the brain.MethodsWe explored whether the anti-cancer drug, Axitinib, a small molecule tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors (VEGFR) can inhibit aberrant cerebrovascular neoangiogenic changes, reduce Aβ deposits and reverse cognitive decline in an animal model of AD. One month post-treatment with Axitinib, we employed a battery of tests to assess cognition and memory in aged Tg2576 AD mice and used molecular analysis to demonstrate reduction of amyloid plaques, BBB leakage, hypervascularity and associated disease pathology.FindingsTargeting the pro-angiogenic pathway in AD using the cancer drug, Axitinib, dramatically reduced cerebrovascular neoangiogenesis, restored BBB integrity, resolved tight-junction pathogenesis, diminishes Aβ depositions in plaques and effectively restores memory and cognitive performance in a preclinical mouse model of AD.InterpretationModulation of neoangiogenesis, in an analogous approach to those used to treat aberrant vascularization in cancer and also in the wet form of age-related macular degeneration (AMD), provides an alternative therapeutic strategy for intervention in AD that warrants clinical investigation.

Fast green FCF inhibits Aβ fibrillogenesis, disintegrates mature fibrils, reduces the cytotoxicity, and attenuates Aβ-induced cognitive impairment in mice

Fast green FCF (FGF) is often used in foods, pharmaceuticals, and cosmetics. However, little is known about the interactions of FGF with amyloid-β protein (Aβ) associated with Alzheimer's disease. In this study, the inhibitory effects of FGF on Aβ fibrillogenesis, the disruption of preformed Aβ fibrils, the reduction of Aβ-induced cytotoxicity, and the attenuation of Aβ-induced learning and memory impairments in mice were investigated. FGF significantly inhibited Aβ fibrillogenesis and disintegrated the mature fibrils as evidenced by thioflavin T fluorescence and atomic force microscopy studies. Co-incubation of Aβ with FGF greatly reduced Aβ-induced cytotoxicity in vitro. Moreover, FGF showed a protective effect against cognitive impairment in Aβ-treated mice. Molecular dynamics simulations further showed that FGF could synergistically interact with the Aβ17-42 pentamer via electrostatic interactions, hydrogen bonds and π-π interactions, which reduced the β-sheet content, and disordered random coils and bend structures of the Aβ17-42 pentamer. This study offers a comprehensive understanding of the inhibitory effects of FGF against Aβ neurotoxicity, which is critical for the search of effective food additives that can combat amyloid-associated disease.

The Prion‐like Cytoplasmic Polyadenylation Element Binding (CPEB) Protein: Can Protein Aggregations in Long‐term Memory Formation inform Memory Loss in Amyloid Associated Neurodegenerative Disease?

Proteinaceous infectious particles or prions are harmful misfolded proteins characterized by a self‐perpetuating amyloidogenic state. These infectious agents target the nervous system and result in rare conditions including Creutzfeldt‐Jacobs disease and kuru, bovine spongiform encephalopathy and scrapie, which are fatal in humans, and cattle and sheep, respectively. Prions have two distinct states: monomeric and amyloidogenic aggregated. The monomeric state is non‐pathogenic while the aggregated state can recruit other monomers into the pathogenic aggregated form. Though not caused by prions, a number of neurodegenerative disorders including Alzheimer’s, Huntington’s, and Parkinson’s disease are associated with the accumulation of misfolded amyloid forming proteins in the brain. In concert with the discoveries involving prions and amyloid‐associated disease, a reductionist approach using the Aplysia sea slug was being developed to study modifications of simple reflexes and underlying neural circuits in an effort to uncover the molecular basis of short‐term and long‐term memory. Through these studies it has been demonstrated that long‐term memory is a function of changes that occur within the synapses and requires synthesis of new proteins in the synapse. More recently, a protein, cytoplasmic polyadenylation element‐binding (CPEB). In contrast to prions, prion‐like proteins are regulated and are functional in their aggregated state. The Blue Valley North 2019–2020 SMART (Students Modeling A Research Topic) Team, supported by the CBM at MSOE, modeled the Apylsia CPEB protein using JMol and 3D printing technology with the goal of exploring this protein’s unique properties and its role in memory formation. In its monomeric state CPEB is inhibited by the microRNA miR22. In contrast, when stimulated by serotonin, a neurotransmitter that induces long‐term synaptic facilitation, miR22 is downregulated, releasing CPEB from its inhibition, and allowing it to enter is active aggregate forming self‐sustaining state. In this state, CPEB activates dormant mRNA located in the axon terminals undergoing long‐term facilitation. Structurally, CPEB contains a N‐terminal region rich in glutamine and asparagine residues, and a C‐terminal RNA binding domain. Neuronal protein homologs with similar N‐terminal regions exist in yeast, mice, and humans as well. It is hypothesized that the N‐terminal region forms both a b‐sheet and an a‐helical coiled coil which facilitate aggregation and the formation of central axis from which the aggregate’s separate RNA binding domains protrude. These open and accessible RNA binding platforms expedite mRNA activation. In this manner CPEB can coordinate the translation of a population of mRNAs important in long‐term memory formation. Though counterintuitive we are hopeful that further structural analysis of the CPEB protein involved in memory formation will inform our understanding of amyloid aggregates associated with memory loss in neurodegenerative disease.

Varicella-Zoster Virus Infection of Primary Human Spinal Astrocytes Produces Intracellular Amylin, Amyloid-β, and an Amyloidogenic Extracellular Environment.

Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid. Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-β [Aβ]) and amyloid, as well as extracellular amylin, Aβ, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aβ42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined. VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aβ, and amyloid. No differences in extracellular amylin, Aβ40, or Aβ42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aβ42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aβ42 aggregation. VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.

Yeast Models for the Study of Amyloid-Associated Disorders and Development of Future Therapy.

First described almost two decades ago, the pioneering yeast models of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, have become well-established research tools, providing both basic mechanistic insights as well as a platform for the development of therapeutic agents. These maladies are associated with the formation of aggregative amyloid protein structures showing common characteristics, such as the assembly of soluble oligomeric species, binding of indicative dyes, and apoptotic cytotoxicity. The canonical yeast models have recently been expanded by the establishment of a model for type II diabetes, a non-neurological amyloid-associated disease. While these model systems require the exogenous expression of mammalian proteins in yeast, an additional amyloid-associated disease model, comprising solely mutations of endogenous yeast genes, has been recently described. Mutated in the adenine salvage pathway, this yeast model exhibits adenine accumulation, thereby recapitulating adenine inborn error of metabolism disorders. Moreover, in line with the recent extension of the amyloid hypothesis to include metabolite amyloids, in addition to protein-associated ones, the intracellular assembly of adenine amyloid-like structures has been demonstrated using this yeast model. In this review, we describe currently available yeast models of diverse amyloid-associated disorders, as well as their impact on our understanding of disease mechanisms and contribution to future potential drug development.

An Imaging and Systems Modeling Approach to Fibril Breakage Enables Prediction of Amyloid Behavior

Delineating the nanoscale properties and the dynamic assembly and disassembly behaviors of amyloid fibrils is key for technological applications that use the material properties of amyloid fibrils, as well as for developing treatments of amyloid-associated disease. However, quantitative mechanistic understanding of the complex processes involving these heterogeneous supramolecular systems presents challenges that have yet to be resolved. Here, we develop an approach that is capable of resolving the time dependence of fibril particle concentration, length distribution, and length and position dependence of fibril fragmentation rates using a generic mathematical framework combined with experimental data derived from atomic force microscopy analysis of fibril length distributions. By application to amyloid assembly of β2-microglobulin in vitro under constant mechanical stirring, we present a full description of the fibril fragmentation and growth behavior, and demonstrate the predictive power of the approach in terms of the samples’ fibril dimensions, fibril load, and their efficiency to seed the growth of new amyloid fibrils. The approach developed offers opportunities to determine, quantify, and predict the course and the consequences of amyloid assembly.

Diagnostic and Therapeutic Potential of Amyloid-Reactive IgG Antibodies Contained in Human Sera

Passive immunotherapy using fibril-reactive mAbs has been shown experimentally to reduce amyloid formation and also accelerate amyloidolysis. We now report that human sera, as well as various sources of pooled human IgG, including pharmacologic formulations of immune globulin i.v. (IGIV), contain Abs that specifically recognize fibrils formed from light chains and other amyloidogenic precursor proteins, including serum amyloid A, transthyretin, islet amyloid polypeptide, and amyloid beta 1-40 peptide, but notably, do not react with these molecules in their native nonfibrillar forms. After isolation of the Abs from IGIV via fibril-conjugated affinity column chromatography, the EC50-binding value for light chains and amyloid beta 1-40 peptide fibrils was approximately 15 nM-a magnitude approximately 200 and 70 times less than that of the unbound fraction and unfractionated product, respectively. Comparable reactivity was found in the case of those formed from serum amyloid A, transthyretin, and islet amyloid polypeptide. The purified Abs immunostained human amyloid tissue deposits and could inhibit fibrillogenesis, as shown in fibril formation and extension assays. Most importantly, in vivo reactivity was evidenced in a murine model when the enriched Abs were used to image amyloid, as well as expedite its removal. These promising experimental results suggest that fibril affinity-purified IGIV has potential as a diagnostic and therapeutic agent for patients with amyloid-associated disease.

Editorial [Hot Topic: Amyloid-Associated Disease Mechanisms in Alzheimers Disease (Guest Editor: Jeroen J.M. Hoozemans)

Editorial [Hot Topic: Amyloid-Associated Disease Mechanisms in Alzheimers Disease (Guest Editor: Jeroen J.M. Hoozemans)

Immunotherapy in systemic primary (AL) amyloidosis using amyloid-reactive monoclonal antibodies.

Heretofore, treatment of patients with primary or light chain-associated (AL) amyloidosis has been directed toward reducing the synthesis of the amyloidogenic precursor protein through conventional or high-dose cytotoxic antiplasma cell chemotherapy. Although such efforts have extended survival, most often the prognosis remains exceedingly poor due to the persistence (or progression) of the pathologic deposits. The development of murine amyloid-reactive monoclonal antibodies (mAbs) has provided another therapeutic approach; namely, passive immunotherapy. These reagents, prepared against human light chain-related fibrils, recognize an epitope common to the beta-pleated structure of AL and other types of amyloid proteins and can effect rapid amyloidolysis when administered to mice injected with human AL amyloid extracts. One such prototypic antibody, the IgG1kappa mAb 11-1F4, has now been chimerized and is undergoing GMP production for an eventual phase I and II clinical trial in patients with AL amyloidosis. Demonstration of the therapeutic efficacy of this amyloid-reactive mAb would provide an important proof-of-principle that this form of immunotherapy also could benefit individuals with other types of inherited or acquired amyloid-associated disease.

Transgenic Mouse Model of AA Amyloidosis

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

Kinetic and Clinical Studies of 2Microglobulin in Continuous Ambulatory Peritoneal Dialysis: Influence of Renal and Enhanced Peritoneal Clearances Using Glucose Polymer

A risk of beta 2-microglobulin (beta 2-M)-associated amyloidosis in long-term CAPD patients has been recognised. We examined the kinetics of beta 2-M and potential clinical manifestations of amyloidosis in patients well-established on CAPD for 1-76 months (mean +/- SEM; 16.4 +/- 14 months). In 57 patients, serum beta 2-M concentration was elevated to 30 +/- 1.8 (mean +/- SEM, mg/l) and correlated positively with the duration on CAPD. In 18 patients studied with variable degrees of residual renal function, serum beta 2-M concentration increased with declining renal function; this was most marked when the creatinine clearance was less than 1 ml/min. Using an isosmolar solution (302 +/- 1.3 mOsm/kg) containing 5% glucose polymer (9.4 mmol/l; MW 16,800), the transperitoneal elimination of beta 2-M was significantly enhanced (1.6 times) compared to conventional 1.36% glucose solution, but without a detectable change in serum concentrations during a 6-h study. No significant difference was found between the estimated minimum volume of distribution of beta 2-M in CAPD and haemodialysis patients. Symptomatic amyloid-associated disease was absent in patients in this study, and may be attributed to the short duration of dialysis.