Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.     

p75 neurotrophin receptor interacts with and promotes BACE1 localization in endosomes aggravating amyloidogenesis

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aβ) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), and this interaction is enhanced in the presence of Aβ. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aβ and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aβ and p75. An increased phosphorylation of APP‐Thr668 and BACE1‐Ser498 by c‐Jun N‐terminal kinase (JNK) in the presence of Aβ and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD.

Cover Image for this Issue: doi. 10.1111/jnc.14163 .

     

Characterization of copper binding to the peptide amyloid-beta(1-16) associated with Alzheimer's disease

Amyloid-beta peptide (Abeta) is the principal constituent of plaques associated with Alzheimer's disease (AD) and is thought to be responsible for the neurotoxicity associated with the disease. Copper binding to Abeta has been hypothesized to play an important role in the neruotoxicity of Abeta and free radical damage, and Cu2+ chelators represent a possible therapy for AD. However, many properties of copper binding to Abeta have not been elucidated clearly, and the location of copper binding sites on Abeta is also in controversy. Here we have used a range of spectroscopic techniques to characterize the coordination of Cu2+ to Abeta(1-16) in solution. Electrospray ionization mass spectrometry shows that copper binds to Abeta(1-16) at pH 6.0 and 7.0. The mode of copper binding is highly pH dependent. Circular dichroism results indicate that copper chelation causes a structural transition of Abeta(1-16). UV-visible absorption spectra suggest that three nitrogen donor ligands and one oxygen donor ligand (3N1O) in Abeta(1-16) may form a type II square-planar coordination geometry with Cu2+. By means of fluorescence spectroscopy, competition studies with glycine and L-histidine show that copper binds to Abeta(1-16) with an affinity of Ka approximately 10(7) M(-1) at pH 7.8. Besides His6, His13, and His14, Tyr10 is also involved in the coordination of Abeta(1-16) with Cu2+, which is supported by 1H NMR and UV-visible absorption spectra. Evidence for the link between Cu2+ and AD is growing, and this work has made a significant contribution to understanding the mode of copper binding to Abeta(1-16) in solution.     

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co‐receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6‐month‐old swedish‐amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose‐ and time‐dependent manner in SH‐SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75^NTR (P75ECD‐FC), or the antibody against the extracellular domain of p75^NTR, blocked the up‐regulation of sortilin induced by Amyloid‐β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH‐SY5Y via the p75^NTR receptor. Inhibition of ROCK, but not Jun N‐terminal kinase, suppressed constitutive and Aβ₄₂‐induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75^NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

     

Molecular dynamics studies of hexamers of amyloid-beta peptide (16-35) and its mutants: influence of charge states on amyloid formation

To study the early stage of amyloid-beta peptide (Abeta) aggregation, hexamers of the wild-type (WT) Abeta(16-35) and its mutants with amyloid-like conformations have been studied by molecular dynamics simulations in explicit water for a total time of 1.7 micros. We found that the amyloid-like structures in the WT oligomers are destabilized by the solvation of ionic D23/K28 residues, which are buried in the fibrils. This means that the desolvation of D23/K28 residues may contribute to the kinetic barrier of aggregation in the early stage. In the E22Q/D23N, D23N/K28Q, and E22Q/D23N/K28Q mutants, hydration becomes much less significant because the mutated residues have neutral amide side-chains. These amide side-chains can form linear cross-strand hydrogen bond chains, or "polar zippers", if dehydrated. These "polar zippers" increase the stability of the amyloid-like conformation, reducing the barrier for the early-stage oligomerization. This is in accord with experimental observations that both the D23/K28 lactamization and the E22Q/D23N mutation promote aggregation. We also found that the E22Q/D23N mutant prefers an amyloid-like conformation that differs from the one found for WT Abeta. This suggests that different amyloid structures may be formed under different conditions.     

Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme

The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.     

Urea-based two-dimensional electrophoresis of beta-amyloid peptides in human plasma: evidence for novel Abeta species

The detailed analysis of beta-amyloid (Abeta) peptides in human plasma is still hampered by the limited sensitivity of available mass spectrometric methods and the lack of appropiate ELISAs to measure Abeta peptides other than Abeta(1-38), Abeta(1-40), and Abeta(1-42). By combining high-yield Abeta immuno- precipitation (IP), IEF, and urea-based Abeta-SDS-PAGE-immunoblot, at least 30 Abeta-immuno-reactive spots were detected in human plasma samples as small as 1.6 mL. This approach clearly resolved Abeta peptides Abeta(1-40), Abeta(1-42), Abeta(1-37), Abeta(1-38), Abeta(1-39), the N-truncated Abeta(2-40), Abeta(2-42), and, for the first time, also Abeta(1-41). Relative quantification indicated that Abeta(1-40) and Abeta(1-42) accounted for less than 60% of the total amount of Abeta peptides in plasma. All other Abeta peptides appear to be either C-terminally or N-terminally truncated forms or as yet uncharacterized Abeta species which migrated as trains of spots with distinct pIs. The Abeta pattern found in cerebrospinal fluid (CSF) was substantially less complex. This sensitive method (2-D Abeta-WIB) might help clarifying the origin of distinct Abeta species from different tissues, cell types, or intracellular pools as well as their amyloidogenicity. It might further help identifying plasma Abeta species suitable as biomarkers for the diagnosis of Alzheimer's disease (AD).     

Clearing the way for tau immunotherapy in Alzheimer's disease

Peptidyl‐prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule‐associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease, the other being amyloid beta (Aβ). PPIases, including Pin1, FK506‐binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline‐directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Aβ production or the toxicity associated with Aβ pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in Alzheimer's disease and represent a family rich in targets for modulating the accumulation and toxicity.

     

Neurotoxicity of Alzheimer's disease A�� peptides is induced by small changes in the A��42 to A��40 ratio

The amyloid peptides Aβ₄₀ and Aβ₄₂ of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ₄₂ seems more pathogenic than Aβ₄₀, the reason for this is not well understood. We show here that small alterations in the Aβ₄₂:Aβ₄₀ ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ₄₂:Aβ₄₀ ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.     

Characterizing bathocuproine self-association and subsequent binding to Alzheimer's disease amyloid beta-peptide by NMR.

Aggregated amyloid beta-peptide (A beta) is the primary constituent of the extracellular plaques and perivascular amyloid deposits associated with Alzheimer's disease (AD). Deposition of the cerebral amyloid plaques is thought to be central to the disease progression. One such molecule that has previously been shown to 'dissolve' deposited amyloid in post-mortem brain tissue is bathocuproine (BC). In this paper 1H NMR chemical shift analysis and pulsed field gradient NMR diffusion measurements were used to study BC self-association and subsequent binding to A beta. The results show that BC undergoes self-association as its concentration increases. The association constant of BC dimerization, Ka, was estimated to be 0.64 mM(-1) at 25 degrees C from 1H chemical shift analysis. It was also found that dimerization of BC appeared to be essential for its binding to A beta. From the self-association constant of BC, Ka, the fraction of dimeric BC in the complex was obtained and the dissociation constant, Kd, of BC bound to A beta40 peptide was then determined to be approximately 1 mM.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Aggregation of Abeta(1-42) in the presence of short peptides: conformational studies

CD and infrared spectroscopic studies were performed on (i) the inhibitory effects of equimolar quantities of LPFFD-OH and LPYFD-NH(2) on the time-dependent aggregation of amyloid beta-protein (Abeta) (1-42) and (ii) the beta-sheet-breaker effects of two-fold molar excess of the pentapeptides on aggregated Abeta(1-42) aged 1 week. The data obtained from the time-dependent studies demonstrated that LPFFD-OH did not significantly influence, whereas LPYFD-NH(2) exerted some inhibitory effect on the aggregation of Abeta(1-42). When added to a solution of Abeta(1-42) aged 1 week, LPFFD-OH accelerated, while LPYFD-NH(2) delayed, but did not prevent further fibrillogenesis. The difference in the effects of these two pentapeptides on the aggregational profile of Abeta(1-42) is probably due to the difference in their conformational preferences: LPFFD-OH adopts a beta-turn and extended structures, while LPYFD-NH(2) adopts a prevailing beta-turn conformation.     

APOE‐modulated Aβ‐induced neuroinflammation in Alzheimer's disease: current landscape,novel data,and future perspective

Chronic glial activation and neuroinflammation induced by the amyloid‐β peptide (Aβ) contribute to Alzheimer's disease (AD) pathology. APOE4 is the greatest AD‐genetic risk factor; increasing risk up to 12‐fold compared to APOE3, with APOE4‐specific neuroinflammation an important component of this risk. This editorial review discusses the role of APOE in inflammation and AD, via a literature review, presentation of novel data on Aβ‐induced neuroinflammation, and discussion of future research directions. The complexity of chronic neuroinflammation, including multiple detrimental and beneficial effects occurring in a temporal and cell‐specific manner, has resulted in conflicting functional data for virtually every inflammatory mediator. Defining a neuroinflammatory phenotype (NIP) is one way to address this issue, focusing on profiling the changes in inflammatory mediator expression during disease progression. Although many studies have shown that APOE4 induces a detrimental NIP in peripheral inflammation and Aβ‐independent neuroinflammation, data for APOE‐modulated Aβ‐induced neuroinflammation are surprisingly limited. We present data supporting the hypothesis that impaired apoE4 function modulates Aβ‐induced effects on inflammatory receptor signaling, including amplification of detrimental (toll‐like receptor 4‐p38α) and suppression of beneficial (IL‐4R‐nuclear receptor) pathways. To ultimately develop APOE genotype‐specific therapeutics, it is critical that future studies define the dynamic NIP profile and pathways that underlie APOE‐modulated chronic neuroinflammation.

     

The ubiquitin proteasomal system: a potential target for the management of Alzheimer's disease

The cellular quality control system degrades abnormal or misfolded proteins and consists of three different mechanisms: the ubiquitin proteasomal system (UPS), autophagy and molecular chaperones. Any disturbance in this system causes proteins to accumulate, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease (AD), Parkinson's disease, Huntington's disease and prion or polyglutamine diseases. Alzheimer's disease is currently one of the most common age‐related neurodegenerative diseases. However, its exact cause and pathogenesis are unknown. Currently approved medications for AD provide symptomatic relief; however, they fail to influence disease progression. Moreover, the components of the cellular quality control system represent an important focus for the development of targeted and potent therapies for managing AD. This review aims to evaluate whether existing evidence supports the hypothesis that UPS impairment causes the early pathogenesis of neurodegenerative disorders. The first part presents basic information about the UPS and its molecular components. The next part explains how the UPS is involved in neurodegenerative disorders. Finally, we emphasize how the UPS influences the management of AD. This review may help in the design of future UPS‐related therapies for AD.     

Raloxifene alleviates amyloid‐β‐induced cytotoxicity in HT22 neuronal cells via inhibiting oligomeric and fibrillar species formation

Raloxifene, a selective estrogen receptor modulator, displays benefits for Alzheimer's disease (AD) prevention in postmenopausal women as hormonal changes during menopause have the potential to influence AD pathogenesis, but the underlying mechanism of its neuroprotection is not entirely clear. In this study, the effects of raloxifene on amyloid‐β (Aβ) amyloidogenesis were evaluated. The results demonstrated that raloxifene inhibits Aβ₄₂ aggregation and destabilizes preformed Aβ₄₂ fibrils through directly interacting with the N‐terminus and middle domains of Aβ₄₂ peptides. Consequently, raloxifene not only reduces direct toxicity of Aβ₄₂ in HT22 neuronal cells, but also suppresses expressions of tumor necrosis factor‐α and transforming growth factor‐β induced by Aβ₄₂ peptides, and then alleviates microglia‐mediated indirect toxicity of Aβ₄₂ to HT22 neuronal cells. Our results suggested an alternative possible explanation for the neuroprotective activity of raloxifene in AD prevention.     

Activation of PKA/SIRT1 signaling pathway by photobiomodulation therapy reduces Aβ levels in Alzheimer's disease models

A hallmark of Alzheimer's disease (AD) is the accumulation of amyloid‐β (Aβ), which correlates significantly with progressive cognitive deficits. Although photobiomodulation therapy (PBMT), as a novel noninvasive physiotherapy strategy, has been proposed to improve neuronal survival, decrease neuron loss, ameliorate dendritic atrophy, and provide overall AD improvement, it remains unknown whether and how this neuroprotective process affects Aβ levels. Here, we report that PBMT reduced Aβ production and plaque formation by shifting amyloid precursor protein (APP) processing toward the nonamyloidogenic pathway, thereby improving memory and cognitive ability in a mouse model of AD. More importantly, a pivotal protein, SIRT1, was involved in this process by specifically up‐regulating ADAM10 and down‐regulating BACE1, which is dependent on the cAMP/PKA pathway in APP/PS1 primary neurons and SH‐SY5Y cells stably expressing human APP Swedish mutation (APPswe). We further found that the activity of the mitochondrial photoacceptor cytochrome c oxidase (CcO) was responsible for PBMT‐induced activation of PKA and SIRT1. Together, our research suggests that PBMT as a viable therapeutic strategy has great potential value in improving cognitive ability and combatting AD.