Amyloid Degradation Research Articles

Amyloid assembly and disassembly.

ABSTRACTAmyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases.

Expression of neprilysin in periodontitis-affected gingival tissues

ObjectiveAlthough the pathogeneses of Alzheimer’s disease (AD) and periodontal diseases have overlapping features, including ageing and chronic inflammation, the association between AD and periodontitis remains unclear. To explore the pathogenesis of periodontitis, a comprehensive gene expression/transcriptome analysis in periodontitis-affected gingival tissues found that the AD pathway was significantly up-regulated in periodontitis-affected gingival tissues. AD-related genes, amyloid beta precursor protein (APP), interleukin-1 beta and compliment 1QA, were significantly elevated in periodontitis. In the present study, balance between mRNA expression of APP and a potent amyloid degradation enzyme, neprilysin (NEP), as well as protein localisation of APP and NEP were analysed. DesignEighteen periodontitis-affected and 18 clinically healthy control gingival tissues were taken from patients with severe chronic periodontitis or undergoing tooth extraction. Total RNA was purified and used for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). The localisation of APP and NEP was analysed by immunohistochemistry (IHC). ResultsBoth APP and NEP genes were up-regulated in periodontitis-affected gingival tissues. APP-expressing macrophages and NEP-expressing neutrophils and fibroblasts, reflecting inflammatory stages, were detected in inflamed gingival tissues by IHC. ConclusionThe up-regulation of APP and NEP mRNA levels in periodontitis-affected gingival tissues compared with healthy controls was confirmed by qRT-PCR analyses. Since NEP is one of the primary enzymes that degrades amyloid beta, increased NEP mRNA levels in periodontitis may act as an inhibitor of amyloid beta accumulation in gingival tissues, balancing increased APP mRNA expression. However, NEP has several effects including degradation of vasoactive substances; therefore, further sresearch is needed.

A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution.

Alzheimer's disease's established primary trigger is β-amyloid (Aβ) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes ( Haass et al., 1992 ; Morel et al., 2013 ; Edgar et al., 2015 ; Ubelmann et al., 2017 ). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons ( Ubelmann et al., 2017 ).

Calycosin improves cognitive function in a transgenic mouse model of Alzheimer's disease by activating the protein kinase C pathway.

The major pathological changes in Alzheimer's disease are beta amyloid deposits and cognitive impairment. Calycosin is a typical phytoestrogen derived from radix astragali that binds to estrogen receptors to produce estrogen-like effects. Radix astragali Calycosin has been shown to relieve cognitive impairment induced by diabetes mellitus, suggesting calycosin may improve the cognitive function of Alzheimer's disease patients. The protein kinase C pathway is upstream of the mitogen-activated protein kinase pathway and exerts a neuroprotective effect by regulating Alzheimer's disease-related beta amyloid degradation. We hypothesized that calycosin improves the cognitive function of a transgenic mouse model of Alzheimer's disease by activating the protein kinase C pathway. Various doses of calycosin (10, 20 and 40 mg/kg) were intraperitoneally injected into APP/PS1 transgenic mice that model Alzheimer's disease. Calycosin diminished hippocampal beta amyloid, Tau protein, interleukin-1beta, tumor necrosis factor-alpha, acetylcholinesterase and malondialdehyde levels in a dose-dependent manner, and increased acetylcholine and glutathione activities. The administration of a protein kinase C inhibitor, calphostin C, abolished the neuroprotective effects of calycosin including improving cognitive ability, and anti-oxidative and anti-inflammatory effects. Our data demonstrated that calycosin mitigated oxidative stress and inflammatory responses in the hippocampus of Alzheimer's disease model mice by activating the protein kinase C pathway, and thereby improving cognitive function.

In vitro and in vivo insulin amyloid degradation mediated by Serratiopeptidase

A transition of amyloidogenic protein by alternative folding pathway under certain conditions leads to the formation of protease resistant amyloid fibrils, having predominantly cross β structure. These amyloids are related to various neurodegenerative diseases and clearance of such amyloids may be a therapeutic approach for amyloid-related diseases. Insulin, that can form amyloids, is widely used as a model amyloidogenic protein for the study of various amyloid related diseases. In this study, insulin amyloids were formed in vitro and the potential of Serratiopeptidase (SP), a fibrinolytic-like serine protease, towards the dissociation of insulin amyloids was explored. The dissociation of the amyloids was demonstrated using in vitro and in vivo using zebrafish model. The amyloid dissociation property was compared with a standard amyloid dissociating enzyme nattokinase (NK). SP shows better amyloid dissociation ability than NK and therefore, SP can be considered as amyloid dissociating agent with potential as a drug candidate for different amyloid related disorders.

Glial alterations from early to late stages in a model of Alzheimer's disease: Evidence of autophagy involvement in Aβ internalization.

Alzheimer's disease (AD) is a progressive neurodegenerative disease without effective therapy. Brain amyloid deposits are classical histopathological hallmarks that generate an inflammatory reaction affecting neuronal and glial function. The identification of early cell responses and of brain areas involved could help to design new successful treatments. Hence, we studied early alterations of hippocampal glia and their progression during the neuropathology in PDAPP-J20 transgenic mice, AD model, at 3, 9, and 15 months (m) of age. At 3 m, before deposits formation, microglial Iba1+ cells from transgenic mice already exhibited signs of activation and larger soma size in the hilus, alterations appearing later on stratum radiatum. Iba1 immunohistochemistry revealed increased cell density and immunoreactive area in PDAPP mice from 9 m onward selectively in the hilus, in coincidence with prominent amyloid Congo red + deposition. At pre-plaque stages, GFAP+ astroglia showed density alterations while, at an advanced age, the presence of deposits was associated with important glial volume changes and apparently being intimately involved in amyloid degradation. Astrocytes around plaques were strongly labeled for LC3 until 15 m in Tg mice, suggestive of increased autophagic flux. Moreover, β-Amyloid fibrils internalization by astrocytes in in vitro conditions was dependent on autophagy. Co-localization of Iba1 with ubiquitin or p62 was exclusively found in microglia contacting deposits from 9 m onward, suggesting torpid autophagy. Our work characterizes glial changes at early stages of the disease in PDAPP-J20 mice, focusing on the hilus as an especially susceptible hippocampal subfield, and provides evidence that glial autophagy could play a role in amyloid processing at advanced stages.

Tyrosine kinase inhibition increases functional parkin‐ B eclin‐1 interaction and enhances amyloid clearance and cognitive performance

Tyrosine kinase inhibitors (TKIs) are effective therapies for leukaemia. Alzheimer is a neurodegenerative disease characterized by accumulation of β-amyloid (plaques) and hyper-phosphorylated Tau (tangles). Here we show that AD animals have high levels of insoluble parkin and decreased parkin-Beclin-1 interaction, while peripheral administration of TKIs, including Nilotinib and Bosutinib, increases soluble parkin leading to amyloid clearance and cognitive improvement. Blocking Beclin-1 expression with shRNA or parkin deletion prevents tyrosine kinase (TK) inhibition-induced amyloid clearance, suggesting that functional parkin-Beclin-1 interaction mediates amyloid degradation. Isolation of autophagic vacuoles (AVs) in AD mouse brain shows accumulation of parkin and amyloid, consistent with previous results in AD brains, while Bosutinib and Nilotinib increase parkin-Beclin-1 interaction and result in protein deposition in the lysosome. These data suggest that decreased parkin solubility impedes parkin-Beclin-1 interaction and amyloid clearance. We identified two FDA-approved anti-cancer drugs as potential treatment for AD.Two FDA-approved tyrosine kinase inhibitor drugs, Bosutinib and Nilotinib, are shown to ameliorate Alzheimer's disease pathology in mouse models by increasing soluble parkin and leading to amyloid clearance and cognitive improvement.

Ketamine reduces amyloid β-protein degradation by suppressing neprilysin expression in primary cultured astrocytes

Pathological accumulation of cortical amyloid β-protein (Aβ) is an early and consistent feature of Alzheimer's disease (AD). Aβ levels in the brain are determined by production and catabolism. Previous studies have suggested that deficits in the brain expression of neprilysin (NEP) and the insulin-degrading enzyme (IDE), which are both proteases involved in amyloid degradation, may promote Aβ deposition in patients with sporadic late-onset AD. Because the incidence of AD increases after surgical intervention, we examined whether ketamine, which is a general anaesthetic with neuroprotective properties for excitotoxic ischaemic damage, is associated with Aβ degradation by inducing NEP and IDE expression. The non-competitive N-methyl-d-aspartate receptor antagonist ketamine and MK801 significantly decreased the expression of NEP, but not IDE, in a concentration- and time-dependent manner through the dephosphorylation of p38 mitogen-activated protein kinase (MAPK) in cultured rat astrocytes. Furthermore, NEP-reduced reagents significantly suppressed the degradation of exogenous Aβ in cultured astrocytes. These results suggested that ketamine suppresses the Aβ degradation of NEP by reducing p38 MAPK-mediated pathway activity.

AA-Amyloid is cleared by endogenous immunological mechanisms

Objective: AA amyloidosis is a complication to longstanding inflammatory diseases, but reduction of amyloid mass has been reported as the inflammation ceases. Not much is known about the endogenous factors that contribute to this amyloid resolution. Herein, we describe the dynamics of amyloid degradation and resolution in experimental murine AA-amyloidosis. Methods: AA-amyloidosis was induced in mice with injections of amyloid enhancing factor (AEF) and by inflammation induced with injections of silver nitrate. Resolution of amyloid deposits was monitored over time. Results: Virtually all amyloid was cleared within 34 weeks. Using the ELISA-technique, antibodies directed against protein AA were detected in animals during amyloid clearance phase and macrophages were shown to internalize amyloid. Also, passive immunization with an amyloid specific monoclonal antibody, produced by a B-cell clone recovered from an animal with advanced AA-amyloidosis, reduced amyloid development in murine AA-amyloidosis. Conclusion: Immunoglobulins co-localize with amyloid deposits and can contribute to amyloid degradation by Fc-receptor mediated phagocytosis, and should be considered key players in the degradation process.

Synaptic localization of acylpeptide hydrolase in adult rat telencephalon

Acylpeptide hydrolase (ACPH), a serine protease present in the central nervous system (CNS), is believed to have a function in modulating synaptic plasticity, cleavage of beta amyloid peptide and degradation of aggregated oxidized proteins. In this report, we demonstrate for the first time the presence of ACPH in the synapse and its preferential localization at the pre-synaptic side. We isolated subcellular fractions from the rat telencephalon enriched in pre- versus post-synaptic components by using differential centrifugation steps to evaluate ACPH catalytic activity and expression level. Relative ACPH levels were determined by Western blot techniques while antibodies against synaptophysin and PSD-95 were used as positive pre- and post-synaptic markers, respectively. Our results show that ACPH protein levels are significantly increased at the synapse, which correlates with a 56% increase in ACPH activity. Furthermore, Western blot experiments show that ACPH is preferentially located at the pre-synaptic side and this is consistent with the increase of its enzymatic activity in fractions enriched in pre-synaptic components. These results give new insights regarding the localization and a putative role of ACPH in the CNS.

Estrogen activates matrix metalloproteinases-2 and -9 to increase beta amyloid degradation

Estrogen is known to affect different aspects of β-amyloid (Aβ) synthesis and degradation. The present work was undertaken to evaluate specifically whether matrix metalloproteinases (MMP) -2 and -9 are involved in Aβ degradation induced by estrogen and whether this is relevant to estrogen-induced neuroprotection. In SH-SY5Y human neuroblastoma cells, 10nM 17β-estradiol (17β-E2) increases mRNA and intracellular protein expression of MMP-2 and -9, as well as the levels of the active forms of both enzymes released in the medium. Specificity of the effect is proved by prevention with the estrogen receptor (ER) antagonist ICI 182,780 (1μM) and involvement of the ERα subtype is confirmed by the use of selective ERα or ERβ agonists (PPT, DPN) and antagonists (MPP, PHTPP). 17β-E2 significantly increases the degradation of Aβ, either transferred with the conditioned medium of H4-APPSw human neuroglioma cells, engineered to overproduce Aβ1–40 and Aβ1–42, or exogenously added as 2μM Aβ1–42. Both these effects are completely prevented by preexposure to the broad spectrum MMP inhibitor GM6001 (5μM). Importantly, the 17β-E2-induced rescue of neuroblastoma cells challenged with 2μM Aβ1–42, an effect prevented by ICI 182,780 (1μM), is mediated by MMPs, as it appears significantly reduced by GM6001 (5μM) as well as by both MMP-2 (200nM) and MMP-9 (200nM) selective inhibitors. In conclusion, the present study shows for the first time that MMP-2 and -9 give a main contribution to estrogen's neuroprotective effect.

Are amyloid‐degrading enzymes viable therapeutic targets in Alzheimer’s disease?

: The amyloid cascade hypothesis of Alzheimer's disease envisages that the initial elevation of amyloid β-peptide (Aβ) levels, especially of Aβ(1-42) , is the primary trigger for the neuronal cell death specific to onset of Alzheimer's disease. There is now substantial evidence that brain amyloid levels are manipulable because of a dynamic equilibrium between their synthesis from the amyloid precursor protein and their removal by amyloid-degrading enzymes (ADEs) providing a potential therapeutic strategy. Since the initial reports over a decade ago that two zinc metallopeptidases, insulin-degrading enzyme and neprilysin (NEP), contributed to amyloid degradation in the brain, there is now an embarras de richesses in relation to this category of enzymes, which currently number almost 20. These now include serine and cysteine proteinases, as well as numerous zinc peptidases. The experimental validation for each of these enzymes, and which to target, varies enormously but up-regulation of several of them individually in mouse models of Alzheimer's disease has proved effective in amyloid and plaque clearance, as well as cognitive enhancement. The relative status of each of these enzymes will be critically evaluated. NEP and its homologues, as well as insulin-degrading enzyme, remain as principal ADEs and recently discovered mechanisms of epigenetic regulation of NEP expression potentially open new avenues in manipulation of AD-related genes, including ADEs.

Genetic Targeting Aromatase in Male Amyloid Precursor Protein Transgenic Mice Down-Regulates β-Secretase (BACE1) and Prevents Alzheimer-Like Pathology and Cognitive Impairment

As brain testosterone plays both androgenic and estrogenic actions due to its conversion into estrogen via aromatase naturally, it is unclear that the age-related reduction of testosterone increased risk of Alzheimer's disease (AD) in men is mediated through androgen alone or both androgen and estrogen mechanisms. Our previous studies using a gene-based approach in mouse model to block the conversion of testosterone into estrogen (aromatase gene knock-out, ArKO), found a depletion of estrogen and increase in testosterone endogenously in males. Here, we use crossing the ArKO mice with APP23 transgenic mice, a mouse model of AD, to produce APP23/Ar(+/-) mice to study the estrogen-independent effect of testosterone on AD. We found a significant reduction in brain plaque formation, improved cognitive function and increase NEP activity in male APP23/Ar(+/-) mice compared with age-matched male APP23 controls. In addition, we found, for the first time, a reduction of beta-secretase (BACE1) enzyme activity, mRNA level and protein expression in the male APP23/Ar(+/-) mice, suggesting that endogenous testosterone, independent from estrogen, may protect against AD in males via two major mechanisms, downregulation of BACE1 activities at transcriptional level to reduce beta amyloid production and upregulation of NEP activities to enhance bate amyloid degradation.

Novel treatments for systemic amyloidosis

Systemic amyloidoses are potentially lethal diseases. Current treatment is directed against an ongoing supply of precursor proteins. Treatment modalities include liver transplantation for transthyretin-related (ATTR) amyloidosis, elimination of inflammation in amyloid A (AA) amyloidosis and intensive chemotherapy in immunoglobulin light chain (AL) amyloidosis. These therapies often elicit substantial morbidity and mortality or insufficient effectiveness. Therefore, the search for new, more effective therapies without serious side-effects remains mandatory. Since cardiac involvement is vital when deciding on therapy, the introduction of N-terminal probrain natriuretic peptide (NT-proBNP) and troponin are of great importance. Determination of serum free light chains has become indispensable in the diagnosis and follow-up of AL amyloidosis. Manipulation of amyloid deposition and degradation might become possible in the future by the use of eprodisate in AA amyloidosis, 4’-iodo-4’-deoxydoxorubicin in AL amylo...

Differential cerebral deposition of IDE and NEP in sporadic and familial Alzheimer's disease

Alzheimer's disease (AD) is characterized by amyloid beta (A beta) accumulation in the brain and is classified as familial early-onset (FAD) or sporadic late-onset (SAD). Evidences suggest that deficits in the brain expression of insulin degrading enzyme (IDE) and neprilysin (NEP), both proteases involved in amyloid degradation, may promote A beta deposition in SAD. We studied by immunohistochemistry IDE and NEP cortical expression in SAD and FAD samples carrying the E280A presenilin-1 missense mutation. We showed that IDE, a soluble peptidase, is linked with aggregated A beta 40 isoform while NEP, a membrane-bound protease, negatively correlates with amyloid angiopathy and its expression in the senile plaques is independent of aggregated amyloid and restricted to SAD cases. NEP, but not IDE, is over-expressed in dystrophic neurites, both proteases are immunoreactive in activated astrocytes but not in microglia and IDE was the only one detected in astrocytes of white matter from FAD cases. Collectively, our results support the notion that gross conformational changes involved in the modification from "natively folded-active" to "aggregated-inactive" IDE and NEP may be a relevant pathogenic mechanism in SAD.

P2-355: Trimer Aβ42 gene vaccine elicits a high level of antibody production when delivered with the GAL4-UAS system and the gene gun

micromolar concentrations. Immunization of young adult mice with hIVIG at 1g/kg i.p. or i.v. resulted in the formation of neutralizing cross-species antibodies as expected, but tolerance developed after both ways of administration in 4-6 weeks. Intraperitoneal administration of hIVIG once weekly for 14 weeks resulted in significant accumulation of human IgG immunoreactivity in the hippocampus of APP/PS1 mice and in some cases opsonization of amyloid plaques. Conclusions: The data suggest that hIVIG contains antibodies that are able to bind Abeta and induce amyloid degradation if provided at high enough concentrations. Development of crossspecies immune response and blood-brain barrier do not prevent testing these effects in transgenic amyloid producing mice also in vivo.

β‐Amyloid Degradation and Alzheimer′s Disease

Extensive β-amyloid (Aβ) deposits in brain parenchyma in the form of senile plaques and in blood vessels in the form of amyloid angiopathy are pathological hallmarks of Alzheimer's disease (AD). The mechanisms underlying Aβ deposition remain unclear. Major efforts have focused on Aβ production, but there is little to suggest that increased production of Aβ plays a role in Aβ deposition, except for rare familial forms of AD. Thus, other mechanisms must be involved in the accumulation of Aβ in AD. Recent data shows that impaired clearance may play an important role in Aβ accumulation in the pathogenesis of AD. This review focuses on our current knowledge of Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), insulin-degrading enzyme (IDE), angiotensin-converting enzyme (ACE), and the plasmin/uPA/tPA system as they relate to amyloid deposition in AD.

Alzheimer's disease: Mechanisms and development of therapeutic strategies

Senile plaques are the most characteristic change in Alzheimer's disease (AD). In senile plaques, β amyloid is deposited, which is composed of aggregated amyloid β protein (Aβ) derived from amyloid precursor protein (APP). Therefore, it is suggested that there exists a mechanism of increase of Aβ production or a decrease of Aβ degradation and/or clearance of β amyloid in AD. Mutations in familial Alzheimer's disease (FAD) genes such as APP, presenilin 1 (PS1) and presenilin 2 (PS2) result in an increase of Aβ production. Apolipoprotein E (ApoE), a genetic risk factor for AD, is involved in Aβ production and/or its clearance. Thus, it is suggested that an inhibition of Aβ production and a facilitation of β amyloid degradation and clearance delay the clinical onset and progression of AD, and it is possible to cure AD even after an onset of the disease, if it is still at an early phase. Researchers studied the fine mechanisms of Aβ production and identified enzymes that cleave‐out Aβ from APP. Inhibitors of the cleaving enzymes are proven to be effective in ameliorating AD‐like conditions in its animal models and are now being applied to humans. Researchers also found an efficient way of clearing β amyloid deposits using the immune system, which was effective in animal models. When it was applied to humans, some patients developed meningoencephalitis as a side‐effect. Therefore, safer vaccines are now being developed. It did not require 20 years for researchers to develop therapeutic strategies since the discovery of Aβ in 1984. Now that AD is becoming a treatable disease, early diagnosis and early treatment will soon become the rule. Notably, AD may not be a psychiatric disorder any more, and mainly neurologists and geriatricians will see patients. Thus, neurogeriatrics will become more and more important.

Extension of Aβ2M amyloid fibrils with recombinant human β2-microglobulin

In order to elucidate the pathogenesis ofAβ2M amyloidosis, we established an experimental system to study the mechanism of amyloid fibril formation or degradation in vitro. We compared the kinetics of Aβ2M amyloid fibril (fAβ2M) extension with native β2microglobulin (n-β2M) purified from the urine of a patient suffering from renal insufficiency, with that with recombinant β2M (r-β2M) in vitro. n-β2M and r-β2M were incubated with fβ32Mpurifiedfrom synovial tissues excised from Aβ2M amyloidosis patients. The fA β2M extension reaction could be explained by a first-order kinetic model in both β2Ms. The extension reaction was greatly dependent on the pH of the reaction mixture and maximum around pH 2.5-3.0 in both p2Ms. ThefAβ2M extended with both p2Ms assumed the similar helical filament structure, although the fibrils extended with r-β2M were slightly wider than those extended with n-β2M and the former fibrils assumed a helical structure more clearly as compared to the latter. In order to obtain pure, unmodifiedfAβ2M, we next extendedfA β2M repeatedly by the algorithmic protocol with r-β2M. As the generation of the extended fibrils proceeded, the initial rate of the extension reaction increased. The ultrastructure of fibrils was completely preserved throughout the repeated extension steps. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that f A P2M extended repeatedly with r-β2Mwere composed solely ofr-β2M. The use of these r-β2M and fAβ2M will be advantageous to assess the effects of several amyloid-associated molecules in the formation or degradation offAβ2M in vitro.

What role(s) may extracellular matrix, particularly heparan sulfate, play in amyloid of Alzheimer's disease

The authors of this article present and attempt to substantiate the thesis that proteoglycan(s), mainly heparan sulfate, are an important ingredient in the pathogenesis of the amyloid found in persons with Alzheimer's disease. Evidence presented indicates that glycosaminoglycans are regular constituents of many amyloid substances including that of senile plaques and congophilic angiopathy of Alzheimer's disease. It is proposed that the proteoglycans play a role in amyloidogenesis by one or a combination of the following mechanisms: 1) inducing amyloid fibrils containing a predominant beta-pleated sheet structure, 2) influencing amyloid deposition to occur at specific tissue sites and/or 3) prevent amyloid degradation.