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Fresh and globular amyloid beta protein (1-42) induces rapid cellular degeneration: evidence for AbetaP channel-mediated cellular toxicity
Abstract
Amyloid beta peptides (AbetaP) deposit as plaques in vascular and parenchymal areas of Alzheimer's disease (AD) tissues and Down's syndrome patients. Although neuronal toxicity is a feature of late stages of AD, vascular pathology appears to be a feature of all stages of AD. Globular and nonfibrillar AbetaPs are continuously released during normal cellular metabolism, form calcium-permeable channels, and alter cellular calcium level. We used atomic force microscopy, laser confocal microscopy, and calcium imaging to examine the real-time and acute effects of fresh and globular AbetaP(1-42), AbetaP(1-40), and AbetaP(25-35) on cultured endothelial cells. AbetaPs induced morphological changes that were observed within minutes after AbetaP treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to AbetaP(1-42). AbetaP(1-42)-induced morphological changes were observed at nanomolar concentrations and were accompanied by an elevated cellular calcium level. Morphological changes were prevented by anti-AbetaP antibody, AbetaP-channel antagonist zinc, and the removal of extracellular calcium, but not by tachykinin neuropeptide, voltage-sensitive calcium channel blocker cadmium, or antioxidants DTT and Trolox. Thus, nanomolar fresh and globular AbetaP(1-42) induces rapid cellular degeneration by elevating intracellular calcium, most likely via calcium-permeable AbetaP channels and not by its interaction with membrane receptors or by activating oxidative pathways. Such rapid degeneration also suggests that the plaques, and especially fibrillar AbetaPs, may not have a direct causative role in AD pathogenic cascades.
... Some HT22 cells treated with 5 M have similar morphology as untreated differentiated cells, these most likely did not have sufficient amyloid accumulation on them to have any detectable effect on cell morphology (Supplementary Figure 15, cell 1). There was a population of HT22 cells, which exhibited contraction along the actin-myosin cytoskeleton during imaging, (Supplementary Figure 15, cell 4), similar to what has been described in an aforementioned report [66]. This contraction along the actin-myosin cytoskeleton was triggered by vertical forces exerted on the cell during AFM imaging, likely as a result of sensitization by A toxicity. ...
... Previous AFM studies of the effects of A 42 on living cells involved acute treatments of less than 3 h with a focus on the effects of A 42 on compressive cellular mechanics [66,101]. They found morphological changes induced by amyloid, in particular, retraction of the cell along the actin-myosin cytoskeleton. ...
... The difference in these two studies could be related to the choice of cell line versus primary cell culture, the choice of probe, and/or force spectroscopy force regime used. The effects of fresh and globular A oligomers on cultured endothelial cells has also been explored by AFM where morphology changes within as early as 30 min following treatment, where cells appear to contract their cytoskeleton under AFM imaging effects which the authors attribute to A ion channels [66]. The effects of A in chronic cell culture models, rather than acute models, has not been explored by AFM imaging in live cell culture. ...
Background:
There is a lack of understanding in the molecular and cellular mechanisms of Alzheimer's disease that has hindered progress on therapeutic development. The focus has been on targeting toxic amyloid-β (Aβ) pathology, but these therapeutics have generally failed in clinical trials. Aβ is an aggregation-prone protein that has been shown to disrupt cell membrane structure in molecular biophysics studies and interfere with membrane receptor signaling in cell and animal studies. Whether the lipid membrane or specific receptors are the primary target of attack has not been determined.
Objective:
This work elucidates some of the interplay between membrane cholesterol and Aβ42 on HT22 neuronal cell viability, morphology, and platelet-derived growth factor (PDGF) signaling pathways.
Methods:
The effects of cholesterol depletion by methyl-β-cyclodextrin followed by treatment with Aβ and/or PDGF-AA were assessed by MTT cell viability assays, western blot, optical and AFM microscopy.
Results:
Cell viability studies show that cholesterol depletion was mildly protective against Aβ toxicity. Together cholesterol reduction and Aβ42 treatment compounded the disruption of the PDGFα receptor activation. Phase contrast optical microscopy and live cell atomic force microscopy imaging revealed that cytotoxic levels of Aβ42 caused morphological changes including cell membrane damage, cytoskeletal disruption, and impaired cell adhesion; cell damage was ameliorated by cellular cholesterol depletion.
Conclusions:
Cholesterol depletion impacted the effects of Aβ42 on HT22 cell viability, morphology, and receptor tyrosine kinase signaling.
... The most representative studies linked to these effects induced by Aβ oligomers are summarized in Table 1. GnRH neuronal cell line [42] cultured endothelial cells [43] bilayer membranes [44,45] ↓ neurotransmitter release hippocampal neurons [46] FzR ↓ Wnt/Fz signaling N2A cells and L-cells [47] mitochondria ↓ complex IV activity APP Tg mice and human brain samples [48] Aβ1-42 oligomers membranes ↑ Ca 2+ lipid vesicles [49] cultured endothelial cells [43] SH-SY5Y cells, oocytes [50,51] hippocampal neurons [52] ↓ axonal transport ↑ non-specific ionic flux neuronal HEK293 membranes [53] ↓ mitochondrial membrane potential hippocampal neurons [54] ↑ oxidative stress IR ↓ activity of IR hippocampal and cortical neurons [55] mGluR/NMDAR ↑ Ca 2+ hippocampal neurons [56] ↑ synaptic glutamate, LTD [57] mGluR ↑ synaptic damage [56] mitochondria ↓ mitochondrial membrane potential APP Tg mice and human brain samples [48] ↑ oxidative stress NMDAR hippocampal neurons [58] p75NTR ↑ NGF-mediated cell death PC12 cells [59] α7/α4β2nAChRs ↑ Ca 2+ hippocampal neurons [60] cortical neurons [61] ↓ surface AMPAR expression hippocampal neurons [60] ↑ endocytosis of NMDAR cortical neurons [61] D1 DAR ↑ epileptic-like activity APP Tg mice [62] Legend: FzR = Frizzled receptors, IR = insulin receptors, mGluR = metabotropic glutamate receptor, NMDAR = NMDA receptor, p75NTR = p75 neurotrophin receptor, nAChRs = nicotinic acetylcholine receptors, D1 DAR = D1 dopamine receptors, GnRH = gonadotropinreleasing hormone, AMPAR = AMPA receptors, APP = amyloid precursor protein, NGF = nerve growth factor. ...
... The most representative studies linked to these effects induced by Aβ oligomers are summarized in Table 1. GnRH neuronal cell line [42] cultured endothelial cells [43] bilayer membranes [44,45] ↓ neurotransmitter release hippocampal neurons [46] FzR ↓ Wnt/Fz signaling N2A cells and L-cells [47] mitochondria ↓ complex IV activity APP Tg mice and human brain samples [48] Aβ1-42 oligomers membranes ↑ Ca 2+ lipid vesicles [49] cultured endothelial cells [43] SH-SY5Y cells, oocytes [50,51] hippocampal neurons [52] ↓ axonal transport ↑ non-specific ionic flux neuronal HEK293 membranes [53] ↓ mitochondrial membrane potential hippocampal neurons [54] ↑ oxidative stress IR ↓ activity of IR hippocampal and cortical neurons [55] mGluR/NMDAR ↑ Ca 2+ hippocampal neurons [56] ↑ synaptic glutamate, LTD [57] mGluR ↑ synaptic damage [56] mitochondria ↓ mitochondrial membrane potential APP Tg mice and human brain samples [48] ↑ oxidative stress NMDAR hippocampal neurons [58] p75NTR ↑ NGF-mediated cell death PC12 cells [59] α7/α4β2nAChRs ↑ Ca 2+ hippocampal neurons [60] cortical neurons [61] ↓ surface AMPAR expression hippocampal neurons [60] ↑ endocytosis of NMDAR cortical neurons [61] D1 DAR ↑ epileptic-like activity APP Tg mice [62] Legend: FzR = Frizzled receptors, IR = insulin receptors, mGluR = metabotropic glutamate receptor, NMDAR = NMDA receptor, p75NTR = p75 neurotrophin receptor, nAChRs = nicotinic acetylcholine receptors, D1 DAR = D1 dopamine receptors, GnRH = gonadotropinreleasing hormone, AMPAR = AMPA receptors, APP = amyloid precursor protein, NGF = nerve growth factor. ...
... In 1993, Arispe and coauthors demonstrated for the first time that Aβ peptides can be incorporated into artificial lipid bilayers, where they are able to form cation-selective channels [44,45]. Since then, evidence have been accumulated in favor of the potential of certain peptides, especially Aβ1-42, to form cation channels in neurons [53,69], oocytes, and endothelial cells [70][71][72][73][74]. Aβ1-40 and Aβ1-42 channels are calcium and zinc permeable [42,43,71,75], and they can profoundly disrupt ionic homeostasis. Aβ1-42 but not Aβ1-40 oligomers also form non-selective cation channels in cellular membranes, which is particularly detrimental for cell signaling [53]. ...
Amyloid-β (Aβ) 1-40 and 1-42 peptides are key mediators of synaptic and cognitive dysfunction in Alzheimer’s disease (AD). Whereas in AD, Aβ is found to act as a pro-epileptogenic factor even before plaque formation, amyloid pathology has been detected among patients with epilepsy with increased risk of developing AD. Among Aβ aggregated species, soluble oligomers are suggested to be responsible for most of Aβ’s toxic effects. Aβ oligomers exert extracellular and intracellular toxicity through different mechanisms, including interaction with membrane receptors and the formation of ion-permeable channels in cellular membranes. These damages, linked to an unbalance between excitatory and inhibitory neurotransmission, often result in neuronal hyperexcitability and neural circuit dysfunction, which in turn increase Aβ deposition and facilitate neurodegeneration, resulting in an Aβ-driven vicious loop. In this review, we summarize the most representative literature on the effects that oligomeric Aβ induces on synaptic dysfunction and network disorganization.
... These articles debate two mechanistic hypotheses. On the one hand, the ''Ab ion channel hypothesis'' suggests that Ab assembles into pore-like structures in lipid membranes, leading to stepwise (or spike-like) fluctuations of transmembrane current that is typical for ion channels (Arispe et al. 1993a(Arispe et al. , b, 1996(Arispe et al. , 2008Durell et al. 1994;Mirzabekov et al. 1994;Kawahara et al. 1996Kawahara et al. , 1997Rhee et al. 1998;Hirakura et al. 1999a, b;Lin et al. 1999Lin et al. , 2001Bhatia et al. 2000;Zhu et al. 2000;Kourie et al. 2001;Kagan et al. 2002Kagan et al. , 2004Kourie et al. 2002;Lin and Kagan 2002;Bahadi et al. 2003;Arispe 2004;Micelli et al. 2004;Quist et al. 2005;Lal et al. 2007;Jang et al. 2008). On the other hand, the ''Ab membrane thinning hypothesis'' postulates a generalized and gradually increasing ion flux as a result of Ab-induced reduction of the dielectric barrier of membranes, for instance, by thinning of membranes Sokolov et al. 2004Sokolov et al. , 2006. ...
... In contrast, when we purged HFIP from Ab samples for 18 h, we found that (1) the residual concentration of HFIP in the Ab samples was consistently below 10 mM and (2) that addition of these Ab samples to membranes (which resulted in a final concentration of HFIP below 0.2 mM) did not result in a gradual increase in ion flux. Instead, we observed in *75% of these experiments a stepwise ion flux as reported previously by several research groups (Fig. 1c) (Arispe et al. 1993a(Arispe et al. , b, 1996(Arispe et al. , 2008Mirzabekov et al. 1994;Kawahara et al. 1996;Rhee et al. 1998;Hirakura et al. 1999a, b;Lin et al. 1999Lin et al. , 2001Bhatia et al. 2000;Kourie et al. 2001;Kagan et al. 2002Kagan et al. , 2004Kourie et al. 2002;Lin and Kagan 2002;Bahadi et al. 2003;Fig. 2 Transmembrane ion flux induced by samples containing welldefined concentrations of HFIP as well as removal of HFIP from Ab samples as a function of the time of purging with nitrogen gas. ...
... The results presented here, along with evidence from other groups (Arispe et al. 1993a(Arispe et al. , b, 1996(Arispe et al. , 2008Mirzabekov et al. 1994;Kawahara et al. 1996Kawahara et al. , 1997Rhee et al. 1998;Hirakura et al. 1999a, b;Lin et al. 1999Lin et al. , 2001Bhatia et al. 2000;Zhu et al. 2000;Kourie et al. 2001Kourie et al. , 2002Kagan et al. 2002Kagan et al. , 2004Lin and Kagan 2002;Bahadi et al. 2003;Arispe 2004;Micelli et al. 2004;Quist et al. 2005;Lal et al. 2007;Jang et al. 2008), clearly demonstrate that Ab peptides are indeed capable of forming ion pores in artificial membranes. They also show that stepwise ion flux is the predominant mode of ion flux across artificial bilayers provided that the samples of Ab are free of organic solvent. ...
... A similar result was obtained by Nagasaka et al. [86] when studying the mechanical properties of the developing cerebral cortical proliferative zone in mice and ferrets, both at the tissue and single-cell level. In 2000, Bhatia et al. [87] applied AFM imaging to demonstrate that morphological changes observed in aged human fibroblasts were a common indicator of cellular degeneration depending on the alloform of induced Aβ oligomers. Lulevich et al. [88] introduced a single-cell compression method where a large colloidal probe is used as the indenter to compress the cell, mimicking the compression of a single cell between two parallel plates. ...
... Bhatia (2000) [87] Aged human fibroblasts Morphological changes in aged human fibroblasts as indicator of cellular degeneration. ...
We review the advances obtained by using Atomic Force Microscopy (AFM)-based approaches in the field of cell/tissue mechanics and adhesion, comparing the solutions proposed and critically discussing them. AFM offers a wide range of detectable forces with a high force sensitivity, thus allowing a broad class of biological issues to be addressed. Furthermore, it allows for the accurate control of the probe position during the experiments, providing spatially resolved mechanical maps of the biological samples with subcellular resolution. Nowadays, mechanobiology is recognized as a subject of great relevance in biotechnological and biomedical fields. Focusing on the past decade, we discuss the intriguing issues of cellular mechanosensing, i.e., how cells sense and adapt to their mechanical environment. Next, we examine the relationship between cell mechanical properties and pathological states, focusing on cancer and neurodegenerative diseases. We show how AFM has contributed to the characterization of pathological mechanisms and discuss its role in the development of a new class of diagnostic tools that consider cell mechanics as new tumor biomarkers. Finally, we describe the unique ability of AFM to study cell adhesion, working quantitatively and at the single-cell level. Again, we relate cell adhesion experiments to the study of mechanisms directly or secondarily involved in pathologies.
... 257,258 Bhatia et al. showed that nanomolar concentrations of globular (nonfibrillar) Aβ 1−40 , Aβ 1−42 , and Aβ 25−35 induced morphological changes leading to cellular degeneration of cultured endothelial cells within minutes and that these cells were the most sensitive to Aβ 1−42 globular oligomers. 259 The mechanism was linked to elevated calcium inside cells, presumably caused by formation of calcium-permeable Aβ 1−42 pores. 259 ...
... 259 The mechanism was linked to elevated calcium inside cells, presumably caused by formation of calcium-permeable Aβ 1−42 pores. 259 ...
... The differential ability of the tested β amyloid peptides to induce platelet adhesive responses in static and flow conditions is extremely interesting, but it remains difficult to explain. Nonetheless, our observations are not isolated, as Aβ1-42 has been identified as the most biologically active of the amyloid peptides [37][38][39]. The biological activity of Aβ1-42 is associated to a marked toxicity of this peptide in several experimental systems [37][38][39]. ...
... Nonetheless, our observations are not isolated, as Aβ1-42 has been identified as the most biologically active of the amyloid peptides [37][38][39]. The biological activity of Aβ1-42 is associated to a marked toxicity of this peptide in several experimental systems [37][38][39]. One possible explanation is the marked propensity of Aβ1-42 to form fibrils compared to Aβ1-40 [40], which is due to the promotion of intermolecular interactions between amyloid monomers induced by the hydrophobic properties of the extra amino acids of Aβ1-42 compared to Aβ1-40. ...
The progression of Alzheimer’s dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β 1-42 (A β 1-42), A β 1-40, and A β 25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to A β 1-42 compared to other A β peptides. In addition, significant platelet spreading was observed over A β 1-42, while A β 1-40, A β 25-35, and the scA β 1-42 control did not seem to induce any platelet spreading, which suggested that only A β 1-42 activates platelet signalling in our experimental conditions. A β 1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other A β peptides did not. The molecular mechanism of A β 1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin α IIb β 3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, A β 1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished A β 1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin α IIb β 3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β 1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer’s patients.
... 19,23−28 The necessary concentrations of peptides used in experiments of ion channel reconstitution to show physiological effects at the cellular level vary significantly, and a high concentration (micromolar) of peptides is usually required. 29 A variety of experimental techniques, including electron microscopy (EM) 20,30,31 and atomic force microscopy (AFM), 18,19,21 have been conducted to investigate the structure of the putative Aβ channels. Most of these studies revealed a βsheet-enriched structure, in which the Aβ peptides assemble to oligomers and these oligomers are then used as subunits to constitute an imperfect annual structure with a central pore. ...
... The most stable Aβ channels and barrels are 16-, 20-, and 24mer oligomers and 12-, 16-, and 20-mer oligomers, respectively, according to these simulations 24,25 and AFM experiments. 18,19,21,26,27 It is proposed that the dimensions of the Aβ channels/barrels should be in a range that allow the formation of interstrand hydrogen bonds between both the Nand C-terminal parts of the β-strands within the subunits in order to maintain the oligomerization of these peptides and provide proper distance for the neighboring subunits to form hydrogen bonds between them and ensure the integrity of the whole channels/barrels. We note that although substantial evidence support the β-sheet-enriched structure for the Aβ channels/barrels mentioned above, 34 molecular modeling has constructed polymorphic pore-like structures constituted by αhelices or combination of helices and β-sheets, 35 which cannot be ruled out with the experimental structures available. ...
Destabilization of cellular ionic homeostasis by toxic β-amyloid (Aβ) channels/barrels, which is a pathogenic mechanism for Alzheimer’s disease (AD), is inhibited by a novel anti-AD drug candidate wgx-50 significantly in our previous biological experiments. In this work, molecular dynamics (MD) simulations are conducted to investigate wgx-50-Aβ channels/barrels interactions, as well as the ion conductance inhibition mechanism. Ion influx from extracellular side to the central pore, which is found in apo-form simulations, is blocked by wgx-50 ligands that bind to the hydrophobic rings at the entrance of the channels/barrels. WGX-50 binding results in smaller pore diameter of the channels/barrels, however the overall morphology of them remains unaffected in accessible simulation time. WGX-50 binding site in this work consists with what we found in our previous simulations of Aβ protofibril. Our work not only investigates the ligand-Aβ channels/barrels interaction mechanism, but also provides insights into rational drug design of Alzheimer’s disease.
... Interaction with amyloid-β, the accumulation of which is associated with Alzheimer's disease, has been shown to induce morphological changes in cell structure, inducing pore-like structures [90]; while these pore-like structures have not been imaged in living neurons, their presence has been supported by the observation of pore-forming oligomers in lipid membrane models [91,92]. In addition to morphological changes, mechanical changes in the cell are critical to cell function. ...
Scanning probe microscopy techniques allow for label-free high-resolution imaging of cells, tissues, and biomolecules in physiologically relevant conditions. These techniques include atomic force microscopy (AFM), atomic force spectroscopy, and Kelvin probe force microscopy (KPFM), which enable high resolution imaging, nanomanipulation and measurement of the mechanoelastic properties of neuronal cells, as well as scanning ion conductance microscopy (SICM), which combines electrophysiology and imaging in living cells. The combination of scanning probe techniques with optical spectroscopy, such as with AFM-IR and tip-enhanced Raman spectroscopy (TERS), allows for the measurement of topographical maps along with chemical identity, enabled by spectroscopy. In this work, we review applications of these techniques to neuroscience research, where they have been used to study the morphology and mechanoelastic properties of neuronal cells and brain tissues, and to study changes in these as a result of chemical or physical stimuli. Cellular membrane models are widely used to investigate the interaction of the neuronal cell membrane with proteins associated with various neurological disorders, where scanning probe microscopy and associated techniques provide significant improvement in the understanding of these processes on a cellular and molecular level.
... In the study's design, Abramov et al. hypothesized that intracellular ion disturbances arise from the appearance of non-selective membrane ion channels formed by beta-amyloid [51]. The formation of such channels was indisputably described by multiple laboratories starting in 1993 [53][54][55][56][57][58][59][60][61][62][63]. In short, adding beta-amyloid to lipid membranes results in forming a non-selective pore, which can pass virtually any cation, including sodium, potassium, and calcium. ...
The manuscript presents the comprehensive integrative theory of the etiology and pathogenesis of Alzheimer’s disease - the amyloid degradation toxicity hypothesis - and describes the logic that underlies it.The analysis of amyloid biomarkers and stable-isotope label kinetics (SILK) studies suggest that AD diagnosis is associated with higher cellular uptake of beta-amyloid. Uptake of beta-amyloid by cells is needed for its cytotoxicity, so the uptake rate should correlate with the rate of neurodegeneration. Also, the initial step in forming extracellular aggregates cannot occur in the interstitial fluid due to the extremely low concentration of beta-amyloid but can occur intralysosomally. Therefore, the density of extracellular aggregates should positively correlate with the rate of cellular amyloid uptake. The model, which considers that both cytotoxicity and aggregation of beta-amyloid are defined by cellular uptake, successfully reproduces the probability distribution of AD diagnosis in the population. Cellular uptake of beta-amyloid is mediated by endocytosis. Endocytosed beta-amyloid induces lysosomal permeabilization that occurs without plasma membrane damage. Lysosomal permeabilization explains ion disturbances, such as an accumulation of intracellular calcium, caused by cell exposure to extracellular beta-amyloid. Some amyloid fragments, produced from beta-amyloid by lysosomal proteases, can form membrane channels in lysosomal membranes, which are large enough to leak cathepsins to the cytoplasm. Appearance of proteases in the cytoplasm results in necrosis and/or initiation of apoptosis. If the cell survives, the damage of lysosomes leads to autophagy failure and slow recycling of mitochondria, promoting the production of reactive oxygen species and potentiating cell damage.Considering the above, the integrative theory of AD etiology and pathogenesis can be formulated. The etiology of AD is the membrane channel formation by amyloid fragments produced in lysosomes. The pathogenesis includes lysosomal permeabilization by giant membrane channels, which leak lysosomal proteases into the cytoplasm. The correlation between the density of amyloid aggregates and the probability of AD appears because the intensity of cellular uptake defines both aggregation rates in vivo and cytotoxicity of beta-amyloid.The amyloid degradation toxicity hypothesis is the integrative theory of Alzheimer’s disease (AD). It successfully interprets multiple phenomena and paradoxes associated with AD pathobiology at various levels, from molecular and cellular to biomarkers. The hypothesis explains the limitations of currently used biomarkers of AD and proposes etiology-related parameters. These parameters could be measured in humans and become novel diagnostic and prognostic clinical tools. Based on the proposed framework, we foresee the development of effective medications to treat, stall the progression of, or prevent disease development.
... On the other hand, Aβ oligomers induce Ca 2+ influx into the cytoplasm. [41][42][43][44] We, therefore, examined Ca 2+ -imaging using a fluorescent dye (fluo-4) and found that plantainoside B suppressed the Aβ-induced Ca 2+ influx in hiPSC-derived cholinergic neurons (Figs. 3G, H). ...
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-β (Aβ) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aβ oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aβ-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aβ aggregates, including its oligomers, using Aβ oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aβ attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aβ injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aβ aggregates including its oligomers and brain tissue from a mouse model of Aβ pathology. In addition, plantainoside B could delay the Aβ aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aβ oligomers, thus interrupting the binding of Aβ oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.
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... The physical and chemical characteristics of Aβ peptides enable the formation of the β-sheet and subsequent aggregation into dimers and, even, large oligomers, which form β-barrel structures for the cation-selective permeability, particularly for Ca 2+ (Figure 3) (Kagan et al., 2002). The nanomole (nM)-level concentrations of Aβ 42 can form Ca 2+ -permeable channels, which elevate [Ca 2+ ] CYT levels and rapidly elicit the degeneration of cultured endothelial cells (Bhatia et al., 2000). When incorporating Aβ 40 into the artificial bilayer membrane, Ca 2+ permeates through the opened Aβ channels (Arispe et al., 1993). ...
Although anything that changes spatiotemporally could be a signal, cells, particularly neurons, precisely manipulate calcium ion (Ca ²⁺ ) to transmit information. Ca ²⁺ homeostasis is indispensable for neuronal functions and survival. The cytosolic Ca ²⁺ concentration ([Ca ²⁺ ] CYT ) is regulated by channels, pumps, and exchangers on cellular membrane systems. Under physiological conditions, both endoplasmic reticulum (ER) and mitochondria function as intracellular Ca ²⁺ buffers. Furthermore, efficient and effective Ca ²⁺ flux is observed at the ER-mitochondria membrane contact site (ERMCS), an intracellular membrane juxtaposition, where Ca ²⁺ is released from the ER followed by mitochondrial Ca ²⁺ uptake in sequence. Hence, the ER intraluminal Ca ²⁺ concentration ([Ca ²⁺ ] ER ), the mitochondrial matrix Ca ²⁺ concentration ([Ca ²⁺ ] MT ), and the [Ca ²⁺ ] CYT are related to each other. Ca ²⁺ signaling dysregulation and Ca ²⁺ dyshomeostasis are associated with Alzheimer’s disease (AD), an irreversible neurodegenerative disease. The present review summarizes the cellular and molecular mechanism underlying Ca ²⁺ signaling regulation and Ca ²⁺ homeostasis maintenance at ER and mitochondria levels, focusing on AD. Integrating the amyloid hypothesis and the calcium hypothesis of AD may further our understanding of pathogenesis in neurodegeneration, provide therapeutic targets for chronic neurodegenerative disease in the central nervous system.
... Caspase-3 is considered the final executor of apoptosis [211] that mediates cytoskeletal and nuclear proteins [212]. Increased Aβ is likely to result in a higher likelihood of oligomer formation, intermediate assemblies, as soluble [213] as well as insoluble aggregates, that possess toxic effects [214][215][216] through several canonical apoptotic pathways, including caspase-3 [217]. Studies have revealed the apoptogenic role of AChE [218], which may lie behind the beneficial role of AChEIs in the early stages of AD [219]. ...
... AFM analysis of A (1-42) aggregates after 12 h was reported by Ruitian Liu et al. [43]. Rajinder Bhatia et al. [44] demonstrated that freshly prepared A (1-42) appear as discrete globular aggregates as imaged by AFM. Neurons examined by SEM 24 hour after addition of the A (1-42) exhibited severe surface blebbing [45]. ...
Keywords: electrochemical impedance spectroscopy (EIS) anodic aluminum oxide (AAO) A-beta (1-42) peptide Alzheimer disease (AD) nanostructured biosensor a b s t r a c t A-beta (1-42) peptide (A (1-42)) is a potential candidate for the prediction of Alzheimer's disease. In this study, we demonstrate a nanostructured biosensor based on electrochemical impedance spectroscopy (EIS) with uniformly deposited gold nanoparticles (GNPs) as the sensing electrode for effective detection of A (1-42). An anodic aluminum oxide (AAO) layer with a nanohemisphere array was used as the substrate. A gold thin film was sputtered onto the AAO substrate to serve as the electrode for GNP deposition and the sensor for A (1-42). A (1-42) antibody was prepared, and its specificity with A (1-42) was verified by Western blot. We observed aggregation of A (1-42) at 1 g ml −1. The morphology of A (1-42) was in the form of round aggregates with diameter of around 1500-2000 nm. EIS measurements for nanostructured biosensors were used to determine the concentration of A (1-42). The plot for the dependence of EIS concentration measurement resulted in an equation Rct = 29098*log [A (1-42)] + 90150 with an R 2 value of 0.9916. The linear detection range was between 1 pg ml −1 and 10 ng ml −1 of A (1-42).
... AFM analysis of A (1-42) aggregates after 12 h was reported by Ruitian Liu et al. [43]. Rajinder Bhatia et al. [44] demonstrated that freshly prepared A (1-42) appear as discrete globular aggregates as imaged by AFM. Neurons examined by SEM 24 hour after addition of the A (1-42) exhibited severe surface blebbing [45]. ...
Keywords: electrochemical impedance spectroscopy (EIS) anodic aluminum oxide (AAO) A-beta (1-42) peptide Alzheimer disease (AD) nanostructured biosensor a b s t r a c t A-beta (1-42) peptide (A (1-42)) is a potential candidate for the prediction of Alzheimer's disease. In this study, we demonstrate a nanostructured biosensor based on electrochemical impedance spectroscopy (EIS) with uniformly deposited gold nanoparticles (GNPs) as the sensing electrode for effective detection of A (1-42). An anodic aluminum oxide (AAO) layer with a nanohemisphere array was used as the substrate. A gold thin film was sputtered onto the AAO substrate to serve as the electrode for GNP deposition and the sensor for A (1-42). A (1-42) antibody was prepared, and its specificity with A (1-42) was verified by Western blot. We observed aggregation of A (1-42) at 1 g ml −1. The morphology of A (1-42) was in the form of round aggregates with diameter of around 1500-2000 nm. EIS measurements for nanostructured biosensors were used to determine the concentration of A (1-42). The plot for the dependence of EIS concentration measurement resulted in an equation Rct = 29098*log [A (1-42)] + 90150 with an R 2 value of 0.9916. The linear detection range was between 1 pg ml −1 and 10 ng ml −1 of A (1-42).
... First, the studies confirmed that the exposure to beta-amyloid increased intracellular calcium levels. However, the authors stressed that the responses were not smooth changes recorded in some studies [9], but consisted of sporadic fluctuations in cytoplasmic calcium similar to the observations of others [8]. These fluctuations looked like waves or oscillations running over minutes, so they occur over a much slower time scale compared to typical calcium changes occurring in these non-excitable cells. ...
In this manuscript, we reassess the data on beta-amyloid-induced changes of intracellular ions concentrations published previously by Abramov et al. (2003, 2004). Their observations made using high-resolution confocal microscopy with fast temporal resolution of images formed by fluorescent ion-sensitive fluorescent probes in living cells represent an unequivocal support for the amyloid channel theory. However, closer look reveals multiple facts which cannot be explained by channel formation in plasma membrane. Recently proposed amyloid degradation toxicity hypothesis provides the interpretation to these facts by considering that channels are formed in the lysosomal membranes.
... In the past few years, in vitro studies demonstrated that the Aβ peptide formed cationselective pores into the plasma membrane, thus causing Ca 2+ influx from the extracellular space across these Aβ pore-channels [161][162][163][164]. However, other in vivo studies showed that Aβ can improve the plasma membrane permeability to both anions and cations by altering its dielectric structure [165]. ...
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that is characterized by amyloid β-protein deposition in senile plaques, neurofibrillary tangles consisting of abnormally phosphorylated tau protein, and neuronal loss leading to cognitive decline and dementia. Despite extensive research, the exact mechanisms underlying AD remain unknown and effective treatment is not available. Many hypotheses have been proposed to explain AD pathophysiology; however, there is general consensus that the abnormal aggregation of the amyloid β peptide (Aβ) is the initial event triggering a pathogenic cascade of degenerating events in cholinergic neurons. The dysregulation of calcium homeostasis has been studied considerably to clarify the mechanisms of neurodegeneration induced by Aβ. Intracellular calcium acts as a second messenger and plays a key role in the regulation of neuronal functions, such as neural growth and differentiation, action potential, and synaptic plasticity. The calcium hypothesis of AD posits that activation of the amyloidogenic pathway affects neuronal Ca2+ homeostasis and the mechanisms responsible for learning and memory. Aβ can disrupt Ca2+ signaling through several mechanisms, by increasing the influx of Ca2+ from the extracellular space and by activating its release from intracellular stores. Here, we review the different molecular mechanisms and receptors involved in calcium dysregulation in AD and possible therapeutic strategies for improving the treatment.
... However, not all patients exhibit such plaques, and many genetic models of AD develop significant neuronal deficits without any histological protein deposits. Also, the addition of already fibrillated Aβ to cells affects cell survival much less than soluble peptide fraction [48]. Therefore, it is quite possible that the plaques are just a consequence of peptide production, but not an actual cause of the disease [31,49], though senile plaques could be a reservoir for the oligomeric peptide [50]. ...
Alzheimer's disease (AD) is the most common cause of dementia and affects millions of people around the world. Neuronal death in AD is initiated by the toxic action of oligomeric amyloid-β (Aβ) peptides. The formation of membrane channels by Aβ is a primary molecular action and does not require any other proteins. Channels are formed by short amyloid fragments faster and more frequently than by full-length peptides. The channel formation is dependent on an electrostatic interaction between a positively charged peptide and a negatively charged membrane. Negative membranes can be found in several locations of a cell – the inner leaflet of plasma membrane, mitochondria, and lysosomes, which all are well-known cellular targets in AD. Considering that the amyloid enters a cell by endocytosis and is exposed to lysosomal enzymes, we propose the amyloid degradation toxicity hypothesis. Endopeptidases degrade the endocytosed peptide. Produced fragments form membrane channels, which can transfer various ions (including protons) and even relatively large compounds. The neutralization of lysosomal content inactivates enzymes, which fails the whole system of recycling cellular content, including autophagy. The permeabilization of lysosomes could also lead to cell death through necrotic and apoptotic mechanisms. We discuss several mechanisms that describe how amyloid degradation products reach plasma and mitochondrial membranes, and form membrane channels. The pathogenesis of AD is discussed at various levels in a context of how the primary molecular mechanism of membrane channel formation could progress into the disease state. The discussion starts at the molecular level and extends to why the development of a disease takes years and is closely associated with aging. The proposed hypothesis offers an interpretation to several clinical observations such as the involvement of iron metabolism and an inverse association between developing Alzheimer's disease and cancer. Predictions about potential biomarkers and effectiveness of future treatments are discussed.
... It has been shown Aβ affects intracellular Ca 2+ concentration by generating Ca 2+ permeable channels in plasma membrane of neurons and astrocytes 70,71 . Works carried out in neuronal cells showed Aβ exposure induced Ca 2+ homeostasis impairments [72][73][74][75] . In vivo, elevated Ca 2+ levels in neuronal cytoplasm were linked to Aβ 42 accumulation 76 . ...
In Alzheimer’s disease (AD) amyloid-β (Aβ) deposits may cause impairments in choroid plexus, a specialised brain structure which forms the blood–cerebrospinal fluid (CSF) barrier. We previously carried out a mass proteomic-based study in choroid plexus from AD patients and we found several differentially regulated proteins compared with healthy subjects. One of these proteins, annexin A5, was previously demonstrated implicated in blocking Aβ-induced cytotoxicity in neuronal cell cultures. Here, we investigated the effects of annexin A5 on Aβ toxicity in choroid plexus. We used choroid plexus tissue samples and CSF from mild cognitive impairment (MCI) and AD patients to analyse Aβ accumulation, cell death and annexin A5 levels compared with control subjects. Choroid plexus cell cultures from rats were used to analyse annexin A5 effects on Aβ-induced cytotoxicity. AD choroid plexus exhibited progressive reduction of annexin A5 levels along with progressive increased Aβ accumulation and cell death as disease stage was higher. On the other hand, annexin A5 levels in CSF from patients were found progressively increased as the disease stage increased in severity. In choroid plexus primary cultures, Aβ administration reduced endogenous annexin A5 levels in a time-course dependent manner and simultaneously increased annexin A5 levels in extracellular medium. Annexin A5 addition to choroid plexus cell cultures restored the Aβ-induced impairments on autophagy flux and apoptosis in a calcium-dependent manner. We propose that annexin A5 would exert a protective role in choroid plexus and this protection is lost as Aβ accumulates with the disease progression. Then, brain protection against further toxic insults would be jeopardised.
... Unlike ion channels, amyloid pores do not have a unitary conductance, they are not gated, (59,60) and have doughnut-like morphologies. (27,48,49,(61)(62)(63) Here, we focus on an oligomer-mediated toxicity mechanism of medin proteins: membrane permeability induced by pore formation. Using biophysical techniques and in silico modeling, we demonstrate that medin induces pore activity in anionic membranes with lipid compositions previously validated as useful model systems of Aβ membrane pore formation. ...
Medin, a 50 amino acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our current results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for pre-aggregated medin species from the growth-phase, and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat-domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy (TEM) images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism (CD) spectroscopy revealed the presence of largely disordered species and suggested the presence of β-sheets. This observation, the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-β fibrils, along with the absence of amyloid fibers in the AFM and TEM images, suggest that medin aggregation into pores follows a non-amyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.
... As occurring during the aging process [40], Aβ peptide disrupts Ca 2+ signalling through several mechanisms, by enhancing the entry of external Ca 2+ and by inducing its release from intracellular stores [44]. Early in vitro studies suggested that Aβ peptides were able to form cation-selective pores into the plasma membrane, thus inducing cytosolic Ca 2+ rises due to Ca 2+ entry across these Aβ pore-channels [45][46][47][48]. However, subsequent in vivo studies demonstrated that exogenously applied Aβ is able to enhance the plasma membrane permeability to both anions and cations by altering its dielectric structure [49]. ...
... Amyloid beta-42 is mainly responsible for formation of insoluble aggregates22. Increase in Aβ are likely to result in a higher likelihood of oligomer formation, intermediate assemblies, such as soluble oligomers of Aβ 23 as well as, insoluble aggregates, possess toxic effects leading to programmed neuronal cell death (apoptosis)[24][25][26], while accumulation of insoluble fibrils of A β peptide, generate negligible neuronal loss27. Accumulation of β-amyloid may eventually lead to neuronal damage and dementia28. ...
To investigate the effect of Curcumin in Celecoxib and STZ induced experimental dementia of Alzheimer disease in mice. Celecoxib and STZ for administrations were used to induce experimental dementia. Curcumin (150 mg/kg, p.o) treatment started two days prior to Celecoxib or STZ administration followed by treatment of Celecoxib (100mg/kg, p.o) and STZ (3mg/kg, i.c.v). Cognitive behaviour was asses by using Morris water maze and elevated plus-maze was used for by measuring transfer latency (TL). On 9 th day animals were sacrificed and acetyl cholinesterase activity was measured to assess cholinergic activity of the brain, thiobarbituric acid reactive species (TBARS) levels, catalase activity, DPPH assay and reduced glutathione (GSH) levels were measured to asses the oxidative stress in brain. The present data demonstrate that curcumin improves memory in celecoxib or STZ induced dementia. From the present data it may be concluded improvement of memory by treatment of Curcumin is due to decrease the oxidative stress.
... A large number of studies have been conducted to elucidate the mechanism of Aβ-induced toxicity in patients. 4,5 The prevailing mechanism for Aβ toxicity includes failure of glutamate uptake, 6 calcium dysregulation, 7 disturbance of intracellular ionic homeostasis, 8 and synaptic dysfunction. 9 The hypothesis on calcium dysregulation is supported by molecular dynamics simulation results 10 and experimental data showing that Aβ forms an ion channel in the membrane. ...
The misfolding of amyloid beta (Aβ) is one of the predominant hallmarks in the pathology of Alzheimer’s disease (AD). In this study, we showed that the formation of Aβ ion channel on the membrane depended on cholesterol concentration. From a mechanical aspect, we found that cholesterol levels affected the stability and assembly of lipid bilayers. Measurements on planar lipid bilayers indicated that a small amount of cholesterol interacted with Aβ proteins and promoted the insertion process. Conversely, high cholesterol integrated the lipid bilayer and exerted an opposite effect on Aβ insertion. Aβ ion channel was then detected by graphene-based field-effect transistors. Results demonstrated that Aβ ion channel promoted Ca2+ flux in the presence of 15% cholesterol but prevent Ca2+ flux in high cholesterol. Thus, cholesterol had a complex impact on Aβ ion channel that can be described as two different effects. First, a small amount of cholesterol interacted with Aβ and facilitated Aβ ion-channel formation in the membrane. Second, a large amount of cholesterol did not induce ion flux in the membrane, which can be explained by the cholesterol damage to the regular distribution of the lipid bilayer. Overall, this study suggested a possible approach to consider cholesterol levels for the treatment of AD patients.
... The coil-helix transition in low-dielectric environments (Fig. 1A-1C) provides a structural rationale for its membrane channel function [116,128,321,322]. Ab is known to form metal ion channels in membranes [116,117,128,130,[323][324][325]. Such interaction involves conformational transition from disordered to helix, as favored upon Cu(II) binding [326] and facilitated by membrane-like micelle environments [122]. ...
In this perspective we list the many clinical, histopathological, genetic and chemical observations relating copper to Alzheimer’s disease (AD). We summarize how the coordination chemistry of the APP/Aβ system is centrally involved in neuronal copper transport at the synapses, and that genetic variations in the gene coding for the copper transporter ATP7B cause a subset of AD, which we call CuAD. Importantly, the distinction between loss of function and gain of toxic function breaks down in CuAD, because copper dyshomeostasis features both aspects directly. We argue that CuAD can be described by a single control variable, a critical, location-dependent copper dissociation constant, Kdc. Loss of functional copper from protein-bound pools reduces energy production and oxidative stress control and is characterized by a reduced pool of divalent Cu(II) with Kd < Kdc. Gain of redox-toxic function is described by more copper with Kd > Kdc. In the blood, the critical threshold is estimated to be Kdc ∼10⁻¹² M whereas at synapses it is argued to be Kdc ∼10⁻⁹ M. The synaptic threshold is close to the values of Kd for Cu(II)-binding to Aβ, prion protein, APP, and α-synuclein, implied in copper buffering at the synapses during glutamatergic transmission. The empirical support for and biochemical and pathological consequences of CuAD are discussed in detail.
... Since the detected ACFs were covered by cellular membrane, the ACFs are generally exhibited as a thick and straight paralleled linear conformation. [44][45][46][47] As a result, the detailed structure information was hard to obtain. In fact, there is little research on the distribution and morphology of ACFs at the nanoscale level, especially in the context of the apical surface of cells, due to its small size, complex topography and soft property at this position. ...
Objectives:
To investigate the heterogeneous feature of actin filaments (ACFs) associated with the cellular membrane in HeLa and HCT-116 cells at the nanoscale level.
Materials and methods:
Fluorescence microscopy coupled with atomic force microscopy (AFM) was used to identify and characterize ACFs of cells. The distribution of ACFs was detected by Fluor-488-phalloidin-labelled actin. The morphology of the ACFs was probed by AFM images. The spatial correlation of the microvilli and ACFs was explored with different forces of AFM loading on cells.
Results:
Intricate but ordered structures of the actin cytoskeletons associated with cellular membrane were characterized and revealed. Two different layers of ACFs with distinct structural organizations were directly observed in HCT-116 and HeLa cells. Bundle-shaped ACFs protruding the cellular membrane forming the microvilli, and the network ACFs underneath the cellular membrane were resolved with high resolution under near-physiological conditions. Approximately 14 nm lateral resolution was achieved when imaging single ACF beneath the cellular membrane. On the basis of the observed spatial distribution of the ultrastructure of the ACF organization, a model for this organization of ACFs was proposed.
Conclusions:
We revealed the two layers of the ACF organization in Hela and HCT-116 cells. The resolved heterogeneous structures at the nanoscale level provide a spatial view of the ACFs, which would contribute to the understanding of the essential biological functions of the actin cytoskeleton.
... It has been shown that certain metal ions, such as Zn(II), may bind to the Aβ aggregates and block pore leakage [57,65]. One in vivo study showed that addition of Zn(II) or removal of Ca(II) ions inhibited Aβ toxicity, while addition of Cd(II) ions (which are known inhibitors of native calcium channels) or antioxidants did not [66,67]. This might be related to Zn(II) but not Cd(II) ions displaying specific binding to the Aβ N-terminal region [18,24], as another study showed that various ions and molecules that interact specifically with the Aβ His13 and His14 residues could block leakage in planar lipid bilayers, and stop cytotoxicity in rat neurons [68], thus indicating similar mechanisms in artificial and biological membranes. ...
The amyloid-β (Aβ) peptides are key molecules in Alzheimer’s disease (AD) pathology. They interact with cellular membranes, and can bind metal ions outside the membrane. Certain oligomeric Aβ aggregates are known to induce membrane perturbations and the structure of these oligomers—and their membrane-perturbing effects—can be modulated by metal ion binding. If the bound metal ions are redox active, as e.g., Cu and Fe ions are, they will generate harmful reactive oxygen species (ROS) just outside the membrane surface. Thus, the membrane damage incurred by toxic Aβ oligomers is likely aggravated when redox-active metal ions are present. The combined interactions between Aβ oligomers, metal ions, and biomembranes may be responsible for at least some of the neuronal death in AD patients.
... Beta amyloid (Ab), a major component of senile plaques, is present in the brains of patients with Alzheimer's disease (AD) and plays a crucial role in AD pathogenesis (Mattson, 2004) (Hardy and Selkoe, 2002). Ab 1-42 contains 42 residues and is closely related to the pathogenesis of AD (Bhatia et al., 2000). Ab 1-42 tends to convert from a native and soluble formation to an insoluble and aggregation statue aggregation, thereby further influencing cell viability. ...
Aβ 1-42 , which is highly toxic to neural cells, is commonly present in the brains of people with Alzheimer's disease. In this study, dynamic changes in cell mechanics were monitored under Aβ-induced toxicity. To investigate the changes in cellular mechanical properties, we used Aβ 1-42 oligomer at different concentrations to treat human neuroblastoma SH-SY5H cells. Results demonstrated a two-stage dynamic change in cell mechanics during neurodegeneration. Additionally, Young's modulus (YM) of the treated cells increased in a short period. The reasons include alteration in surface tension, osmotic pressure, and actin polymerization. Rough cellular membranes were observed from atomic force microscope (AFM) measurement. However, the cellular YM gradually decreased when the cells were continuously exposed to Aβ 1-42 or to a high concentration of Aβ 1-42 . The major reason for the decreased YM was microtubule disassembly. Dynamic change in YM reflects different activities in cytoplasm in response to Aβ 1-42 . The characteristic changes in cell mechanics provided insights into the dynamic neurodegeneration process of cells induced by Aβ 1-42 oligomer.
... Additionally, free radicals are reported to enhance membrane disruption, which further amplifies the unregulated calcium influx and finally leads to AD. Furthermore, as documented in different experiments and molecular dynamics simulation studies [14,15,16], formation of ion channels by Ab-42 enhances the degenerative process of AD. ...
Numerous studies have reported that amyloid-beta 42 (Ab-42) protein is a high-profile risk factor associated with the onset and progression of Alzheimer’s disease (AD). Accumulation of extracellular senile plaques, synaptic degeneration, and intracellular neurofibrillary tangles were recorded as essential features that facilitate the onset of Ab-42, resulting in AD. Hence, we attempted a new screening technique to discover potential inhibitors against Ab-42 using an in silico deep neural network approach. We screened PubChem compounds library and found wgx-50 as a potential inhibitor of Ab-42. Also, synergistic effects of wgx-50–gold nanoparticles (AuNPs) complex induced significant inhibition of Ab-42, compared with those of wgx-50 alone. Further, molecular docking analysis, systems biology approach, and time course simulation confirmed that synergistic effects of wgx-50–AuNPs complex have potential application in the treatment for AD. Additionally, we proposed the biological circuit for AD induced by Ab-42 that can be used to monitor the effect of drugs on AD.
... It has been shown monomeric Aβ is not sufficient to be toxic, however, the aggregation state of Aβ is crucial to exert its neurotoxic effects [36][37][38]. Aβ aggregates can disrupt Ca 2+ signaling in several ways as it can trigger Ca 2+ release from ER stores through the InsP 3 R and RyR [39,40] and allow Ca 2+ influx by forming cation permeation pores on the plasma membrane [41,42]. Conversely, emerging data demonstrated that cellular Ca 2+ dysregulation can affect APP processing. ...
Alzheimer's disease (AD) is the most common type of dementia and is characterized by the accumulation of amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Much attention has been given to develop AD treatments based on the amyloid cascade hypothesis; however, none of these drugs had good efficacy at improving cognitive functions in AD patients suggesting that Aβ might not be the disease origin. Thus, there are urgent needs for the development of new therapies that target on the proximal cause of AD. Cellular calcium (Ca2+) signals regulate important facets of neuronal physiology. An increasing body of evidence suggests that age-related dysregulation of neuronal Ca2+ homeostasis may play a proximal role in the pathogenesis of AD as disrupted Ca2+ could induce synaptic deficits and promote the accumulation of Aβ plaques and neurofibrillary tangles. Given that Ca2+ disruption is ubiquitously involved in all AD pathologies, it is likely that using chemical agents or small molecules specific to Ca2+ channels or handling proteins on the plasma membrane and membranes of intracellular organelles to correct neuronal Ca2+ dysregulation could open up a new approach to AD prevention and treatment. This review summarizes current knowledge on the molecular mechanisms linking Ca2+ dysregulation with AD pathologies and discusses the possibility of correcting neuronal Ca2+ disruption as a therapeutic approach for AD.
... The production of beta-amyloid (Aβ)−an Alzheimer's disease biomarker−results from the amyloidogenic processing of the amyloid precursor protein (APP) (7,8), which is a ubiquitously expressed transmembrane glycoprotein (9,10). Physiologically, Aβ is released continuously in soluble globular form as a product of whole body cellular metabolism, and it circulates in the bloodstream (11). The Aβ generated in the brain can be eliminated from the brain by the BBB through receptor-mediated transport and perivascular drainage via the vascular basement membrane (12,13). ...
Objective
Whether blood-brain barrier (BBB) disruption induced by chronic spontaneous hypertension is associated with beta-amyloid (Aβ) accumulation in the brain remains poorly understood. The purpose of this study was to investigate the relationship between BBB disruption and Aβ influx and accumulation in the brain of aged rats with chronic spontaneous hypertension.
Materials and Methods
Five aged spontaneously hypertensive rats (SHRs) and five age-matched normotensive Wistar-Kyoto (WKY) rats were studied. The volume transfer constant (Ktrans) obtained from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to evaluate BBB permeability in the hippocampus and cortex in vivo. The BBB tight junctions, immunoglobulin G (IgG), Aβ, and amyloid precursor protein (APP) in the hippocampus and cortex were examined with immunohistochemistry.
Results
As compared with WKY rats, the Ktrans values in the hippocampus and cortex of the SHRs increased remarkably (0.316 ± 0.027 min⁻¹ vs. 0.084 ± 0.017 min⁻¹, p < 0.001 for hippocampus; 0.302 ± 0.072 min⁻¹ vs. 0.052 ± 0.047 min⁻¹, p < 0.001 for cortex). Dramatic occludin and zonula occludens-1 losses were detected in the hippocampus and cortex of SHRs, and obvious IgG exudation was found there. Dramatic Aβ accumulation was found and limited to the area surrounding the BBB, without extension to other parenchyma regions in the hippocampus and cortex of aged SHRs. Alternatively, differences in APP expression in the hippocampus and cortex were not significant.
Conclusion
Blood-brain barrier disruption is associated with Aβ influx and accumulation in the brain of aged rats with chronic spontaneous hypertension. DCE-MRI can be used as an effective method to investigated BBB damage.
... It has been shown Aβ affects intracellular Ca 2+ concentration by generating Ca 2+ permeable channels in plasma membrane of neurons and astrocytes 70,71 . Works carried out in neuronal cells showed Aβ exposure induced Ca 2+ homeostasis impairments [72][73][74][75] . In vivo, elevated Ca 2+ levels in neuronal cytoplasm were linked to Aβ 42 accumulation 76 . ...
... For another set of experiments, 8 9 10 5 cells were plated and differentiated with RA (100 nM) for 24 hrs. Differentiated SH-SY5Y cells were pre-treated for 2 hrs with DMF 1, 10, 30 lM, respectively; then, SH-SY5Y cells were stimulated with Ab 1-42 1 lM for 24, as previously described [32], for Western blot analysis and biochemical assay. ...
Alzheimer disease (AD) is characterized by a complex heterogeneity of pathological changes, and any therapeutic approach categorically requires a multi-targeted way. It has been demonstrated that together with the hallmarks of the disease such as neurofibrillary tangles and senile plaques, oxidative and inflammatory stress covered an important role. Dimethyl fumarate (DMF) is an orally bioavailable methyl ester of fumaric acid and activator of Nrf2 with potential neuroprotective and immunomodulating activities. Therefore, the aim of the present work was to evaluate the potential beneficial effects of DMF, compared with its active metabolite monomethyl fumarate (MMF) (both at 30 μM) in an in vitro Alzheimer's model using SH-SY5Y human neuroblastoma cell lines stimulated with amyloid-beta (Aβ). Moreover, the effect of DMF, compared with MMF, was evaluate by an ex vivo model using organotypic hippocampal slice cultures stimulated with Aβ1-42 (1 μg/ml), to better understand its action in a pathological setting. In both models, DMF pre-treatment (30 μM) preserved cellular viability from Aβ stimulation, reducing tau hyper-phosphorylation, much more efficiently then MMF (30 μM). Moreover, DMF was able to induce an activation of manganese superoxide dismutase (MnSOD) and heme-oxygenase-1 (HO-1), decreasing the severity of oxidative stress. Our results showed important multi-protective effects of DMF pre-treatment from Aβ stimulation both in in vitro and ex vivo models, highlighting an Nrf2/NF-κB-dependent mechanism, which could provide a valuable support to the therapies for neurodegenerative diseases today.
... However, the exact disease mechanism has not yet been fully elucidated. A prevailing mechanism of AD pathology postulates that Aβ oligomers negatively affect neuronal function and survival by forming ion permeable pores, resulting in the destabilization of cell ionic homeostasis [8][9][10][11][12] . This hypothesis is supported by data from several experimental techniques and molecular dynamics (MD) simulations studying AβEinduced permeability of ions across model lipid membranes 9,10,13,14 , as well as an "optical patch clamp" method whereby total internal reflection fluorescence (TIRF) microscopy data revealed the presence of localized Ca 2+ transients during cellular influx across Xenopus oocyte membranes 15 . ...
Amyloid-β (Aβ) oligomers are the predominant toxic species in the pathology of Alzheimer's disease. The prevailing mechanism for toxicity by Aβ oligomers includes ionic homeostasis destabilization in neuronal cells by forming ion channels. These channel structures have been previously studied in model lipid bilayers. In order to gain further insight into the interaction of Aβ oligomers with natural membrane compositions, we have examined the structures and conductivities of Aβ oligomers in a membrane composed of brain total lipid extract (BTLE). We utilized two complementary techniques: atomic force microscopy (AFM) and black lipid membrane (BLM) electrical recording. Our results indicate that Aβ1-42 forms ion channel structures in BTLE membranes, accompanied by a heterogeneous population of ionic current fluctuations. Notably, the observed current events generated by Aβ1-42 peptides in BTLE membranes possess different characteristics compared to current events generated by the presence of Aβ1-42 in model membranes comprised of a 1:1 mixture of DOPS and POPE lipids. Oligomers of the truncated Aβ fragment Aβ17-42 (p3) exhibited similar ion conductivity behavior as Aβ1-42 in BTLE membranes. However, the observed macroscopic ion flux across the BTLE membranes induced by Aβ1-42 pores was larger than for p3 pores. Our analysis of structure and conductance of oligomeric Aβ pores in a natural lipid membrane closely mimics the in vivo cellular environment suggesting that Aβ pores could potentially accelerate the loss of ionic homeostasis and cellular abnormalities. Hence, these pore structures may serve as a target for drug development and therapeutic strategies for AD treatment.
... It was suggested in the amyloid cascade hypothesis that fibrillar forms of A, deposited in amyloid plaques, were responsible for neuronal dysfunction [12]. Today, soluble and diffusible A oligomers are known to be the main synaptotoxic and proapoptotic species, inducing learning deficits and neuronal loss as well as inhibition of long-term potentiation [13][14][15][16]. ...
Protein misfolding and aggregation are fundamental features of the majority of neurodegenerative diseases, like Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, and prion diseases. Proteinaceous deposits in the brain of the patient, e.g., amyloid plaques consisting of the amyloid-β (Aβ) peptide and tangles composed of tau protein, are the hallmarks of AD. Soluble oligomers of Aβ and tau play a fundamental role in disease progression, and specific detection and quantification of the respective oligomeric proteins in cerebrospinal fluid may provide presymptomatically detectable biomarkers, paving the way for early diagnosis or even prognosis. Several studies on the development of techniques for the specific detection of Aβ oligomers were published, but some of the existing tools do not yet seem to be satisfactory, and the study results are contradicting. The detection of oligomers is challenging because of their polymorphous and unstable nature, their low concentration, and the presence of competing proteins and Aβ monomers in body fluids. Here, we present an overview of the current state of the development of methods for Aβ oligomer specific detection and quantitation. The methods are divided in the three subgroups: (i) enzyme linked immunosorbent assays (ELISA), (ii) methods for single oligomer detection, and (iii) others, which are mainly biosensor based methods.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia and memory loss in the elderly population. The amyloid-β peptide (Aβ) is one of the main pathogenic factors in AD and is known to cause damage to neuronal cellular membranes. There is no cure currently available for AD, and new approaches, including preventive strategies, are highly desirable. In this work, we explore the possibility of protecting neuronal membranes from amyloid-induced damage with naturally existing sugar trehalose. Trehalose has been shown to protect plant cellular membranes in extreme conditions and modify Aβ misfolding. We hypothesize that trehalose can protect the neuronal membrane from amyloid toxicity. In this work, we studied the protective effect of trehalose against Aβ1-42-induced damage in model lipid membranes (DPPC/POPC/Cholesterol) using atomic force microscopy (AFM) and black lipid membrane (BLM) electrophysiology. Our results demonstrated that Aβ1-42 damaged membranes and led to ionic current leakage across these membranes due to the formation of various defects and pores. The presence of trehalose reduced the ion current across membranes caused by Aβ1-42 peptide damage, thus efficiently protecting the membranes. These findings suggest the trehalose sugar can potentially be useful in protecting neuronal membranes against amyloid toxicity in AD.
Results from recent clinical trials of antibodies that target amyloid-β (Aβ) for Alzheimer’s disease have created excitement and have been heralded as corroboration of the amyloid cascade hypothesis. However, while Aβ may contribute to disease, genetic, clinical, imaging and biochemical data suggest a more complex aetiology.
Here we review the history and weaknesses of the amyloid cascade hypothesis in view of the new evidence obtained from clinical trials of anti-amyloid antibodies. These trials indicate that the treatments have either no or uncertain clinical effect on cognition. Despite the importance of amyloid in the definition of Alzheimer’s disease, we argue that the data point to Aβ playing a minor aetiological role.
We also discuss data suggesting that the concerted activity of many pathogenic factors contribute to Alzheimer’s disease and propose that evolving multi-factor disease models will better underpin the search for more effective strategies to treat the disease.
In Alzheimer's disease (AD), secretion and deposition of amyloid beta peptides (Aβ) have been associated with blood-brain barrier dysfunction. However, the role of Aβ in endothelial cell (EC) dysfunction remains elusive. Here we investigated AD mediated EC activation by studying the effect of Aβ secreted from human induced pluripotent stem cell-derived cortical neurons (hiPSC-CN) harboring a familial AD mutation (Swe+/+) on human brain microvascular endothelial cells (HBMECs) in 2D and 3D perfusable microvessels. We demonstrated that increased Aβ levels in Swe+/+ conditioned media (CM) led to stress fiber formation and upregulation of genes associated with endothelial inflammation and immune-adhesion. Perfusion of Aβ-rich Swe+/+ CM induced acute formation of von Willebrand factor (VWF) fibers in the vessel lumen, which was attenuated by reducing Aβ levels in CM. Our findings suggest that Aβ peptides can trigger rapid inflammatory and thrombogenic responses within cerebral microvessels, which may exacerbate AD pathology.
The discovery of effective therapeutics targeting amyloid-β (Aβ) aggregates for Alzheimer's disease (AD) has been very challenging, which suggests its complicated etiology associated with multiple pathogenic elements. In AD-affected brains, highly concentrated metals, such as copper and zinc, are found in senile plaques mainly composed of Aβ aggregates. These metal ions are coordinated to Aβ and affect its aggregation and toxicity profiles. In this review, we illustrate the current view on molecular insights into the assembly of Aβ peptides in the absence and presence of metal ions as well as the effect of metal ions on their toxicity.
In this manuscript, we reassess the data on beta-amyloid-induced changes of intracellular ions concentrations published previously by Abramov et al. (2003, 2004). Their observations made using high-resolution confocal microscopy with fast temporal resolution of images formed by fluorescent ion-sensitive fluorescent probes in living cells represent an unequivocal support for the amyloid channel theory. However, closer look reveals multiple facts which cannot be explained by channel formation in plasma membrane. Recently proposed amyloid degradation toxicity hypothesis provides the interpretation to these facts by considering that channels are formed in the lysosomal membranes.
Aβ deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aβ1–40 and Aβ1–42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aβ variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aβ4–42 exhibits a high tendency to form β-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aβ4–42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aβ4–42. An Aβ variant with a double truncation at phenylalanine-4 and leucine 34, (Aβ4–34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aβ species in AD pathophysiology.
The strength and efficiency of synaptic connections are affected by the environment or the experience of the individual. This property, called synaptic plasticity, is directly related to memory and learning processes and has been modeled at the cellular level. These types of cellular memory and learning models include specific stimulation protocols that generate a long-term strengthening of the synapses, called long-term potentiation, or a weakening of the said long-term synapses, called long-term depression. Although, for decades, researchers have believed that the main cause of the cognitive deficit that characterizes Alzheimer's disease (AD) and aging was the loss of neurons, the hypothesis of an imbalance in the cellular and molecular mechanisms of synaptic plasticity underlying this deficit is currently widely accepted. An understanding of the molecular and cellular changes underlying the process of synaptic plasticity during the development of AD and aging will direct future studies to specific targets, resulting in the development of much more efficient and specific therapeutic strategies. In this review, we classify, discuss, and describe the main findings related to changes in the neurophysiological mechanisms of synaptic plasticity in excitatory synapses underlying AD and aging. In addition, we suggest possible mechanisms in which aging can become a high-risk factor for the development of AD and how its development could be prevented or slowed.
A major hallmark of Alzheimer's disease (AD) is the accumulation and deposition of fibrillar aggregates of the amyloid-β (Aβ) peptide into neuritic plaques. These amyloid deposits were thought to play a central role in AD; however, the correlation between plaque load and disease is weak. Increasing evidence supports the notion that a variety of small, globular aggregates of Aβ, referred to broadly as Aβ oligomers (AβO), may in fact be the primary culprits associated with neurotoxicity. Evaluation of AβO structure and physiological activity is complicated by their metastability, heterogeneity, complex aggregation pathways, and dependence on experimental conditions. Numerous different types of oligomers have been reported, and these have been associated with varying degrees of toxicity and modes of interaction. Here, we briefly review AβOs with a focus on their formation, structure, and biophysical methods applied to their investigation.
Human amyloidβ1-42 (hAβ1-42) peptides are known to self-aggregate into oligomers that contribute to the degeneration of neurons and development of Alzheimer's disease (AD) pathology. Unlike humans, rodents do not develop AD, possibly due to differences in three amino acids (R5G, Y10F and H13R) within the hydrophilic N-terminal domain of Aβ1-42. This is partly supported by evidence that hAβ1-42 is more prone to fibrillization and has a higher cellular toxicity than rodent Aβ1-42 (rAβ1-42). Mutagenesis studies, however, have shown that correlation between fibrillization potential and toxicity is not always direct. Thus, to understand better how N-terminal mutations can affect hAβ1-42 toxicity through oligomerization, we evaluated fibrillization kinetics, oligomer sizes and toxicity profiles of double mutant (human towards rodent) Aβ1-42. Additionally, we tested the mutant peptides in combination with hAβ1-42, to assess effects on hAβ1-42 aggregation/toxicity. Our results clearly show that double mutations to humanize rAβ1-42 result in a significantly reduced efficiency of fibril formation, as determined by Thioflavin-T aggregation assays and confirmed with electron micrographic studies. Interestingly, the mutants are still able to aggregate into oligomers, which are predominantly larger than those comprised of hAβ1-42. Our cell viability experiments further showed a rank order of oligomer toxicity of hAβ1-42>rAβ1-42>mutant Aβ1-42, suggesting that toxicity can be influenced by N-terminal Aβ1-42 mutations via reduction of fibril formation and/or alteration of oligomer size. These results, taken together, confirm that N-terminal mutations can affect Aβ fibril and oligomer formation with reduced toxicity despite lying outside the core amyloid region of Aβ peptide.
Lipid membranes play a fundamental role in the pathological development of protein misfolding diseases. Several pieces of evidence suggest that the lipid membrane could act as a catalytic surface for protein aggregation. Furthermore, a leading theory indicates the interaction between the cell membrane and misfolded oligomer species as the responsible for cytotoxicity, hence, for neurodegeneration in disorders such as Alzheimer's and Parkinson's disease. The definition of the mechanisms that drive the interaction between pathological protein aggregates and plasma membrane is fundamental for the development of effective therapies for a large class of diseases. Atomic force microscopy (AFM) has been employed to study how amyloid aggregates affect the cell physiological properties. Considerable efforts were spent to characterize the interaction with model systems, i.e., planar supported lipid bilayers, but some works also addressed the problem directly on living cells. Here, an overview of the main works involving the use of the AFM on both model system and living cells will be provided. Different kind of approaches will be presented, as well as the main results derived from the AFM analysis.
Alzheimer’s disease is increasingly recognized to be linked to the function and status of metal ions, and recently, the amyloid hypothesis has been strongly intertwined with the metal ion hypothesis; in fact, these two hypotheses fit well together and are not mutually contradictory. This review focuses on the essential coordination chemistry and biochemistry that relate transition metal ions iron, copper, and zinc to β-amyloid (Aβ) and most likely define the peptide's roles in neurons. The metal-Aβ interactions have elements of both gain of toxic function, as usually considered, but also loss of natural functions, as emphasized in this review. Both these aspects and their relationships are discussed and their implications for future therapeutic strategies are outlined.
Copper is vital to normal brain function; but its potent redox activity demands tight regulation to maintain the integrity of copper homeostasis. Disrupted regulation can result in copper displacement, causing inadvertent interactions between copper and cellular components, which can enhance the production of reactive oxygen species (ROS), formation of neurotoxic copper–protein aggregates, and eventually, neuronal cell death. Disrupted copper homeostasis is a feature common to many neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease, Wilson’s disease, Menkes disease and prion disease. This review focuses on the involvement of copper in AD. An intrinsic reciprocal relationship exists between copper and AD-associated proteins, amyloid precursor protein (APP) and BACE1. Under conditions of copper dysregulation, the postsynaptic release of both copper and Aβ into the synaptic cleft of glutamatergic neurons promotes the abnormal interaction of redox-active Aβ with copper, forming neurotoxic soluble Aβ oligomers. A cascade of Aβ aggregation ensues, resulting in extracellular amyloid plaques, a pathological hallmark of AD. Additionally, copper also participates in the aggregation of tau, the core component of neurofibrillary tangles, which is the other defining pathology of AD brains. Therapeutic strategies targeting interactions among Aβ, tau and metals to restore copper and metal balance have disease-modifying promise.
Alzheimer's disease (AD) is a neurodegenerative disorder with the histopathological hallmark of extracellular accumulation of amyloid-β (Aβ) peptide in brain senile plaques. Though many studies have shown the neural toxicity from various forms of Aβ peptides, the subcellular mechanisms of Aβ peptide are still not well understood, partially due to the technical challenges of isolating axons or dendrites from the cell body for localized investigation. In this study, the subcellular toxicity and localization of Aβ peptides are investigated by utilizing a microfluidic compartmentalized device, which combines physical restriction and chemotactic guidance to enable the isolation of axons and dendrites for localized pharmacological studies. It is found that Aβ peptides induced neuronal death is mostly resulted from Aβ treatment at cell body or axonal processes, but not at dendritic neurites. Simply applying Aβ to axons alone induces significant hyperactive spiking activity. Dynamic transport of Aβ aggregates is only observed between axon terminal and cell body. In addition to differential cellular uptake, more Aβ-peptide secretion is detected significantly from axons than from dendritic side. These results clearly demonstrate the existence of a localized mechanism in Aβ-induced neurotoxicity, and can potentially benefit the development of new therapeutic strategies for AD.
Amyloid precursor protein (APP) is evolutionary conserved protein expressed in endothelial cells of cerebral and peripheral arteries. In this review, we discuss mechanisms responsible for expression and proteolytic cleavage of APP in endothelial cells. We focus on physiological and pathological implications of APP expression in vascular endothelium.
Amyloid-β (Aβ) is widely recognized as toxic to neuronal cells. Its deposition on plasma and intracellular membranes and aggregation into amyloid plaques can disturb the composition and physiological function of neurons. Whether a physical property of cells, such as stiffness, is altered by endogenously overexpressed Aβ has not yet been investigated. In this study, we used human neuroblastoma cells stably overexpressing amyloid precursor protein (APP) and its Swedish mutant form (APPswe) to measure the changes in cell stiffness. Our results showed that the stiffness of cells overexpressing APP or APPswe was higher than that of control SH-SY5Y cells. Either reducing levels of Aβ with the γ secretase inhibitor DAPT or blocking the membrane calcium channel formed by Aβ with tromethamine decreased cell stiffness to a level close to the control SH-SY5Y cells. Our results suggested that Aβ, not APP, contributed to increased cell stiffness and that closure of calcium channels formed by Aβ can alleviate the effects of Aβ on membrane stiffness.
The inability to effectively halt or cure Alzheimer's disease (AD), exacerbated by the recent failures of high-profile clinical trials, emphasizes the urgent need to understand the complex biochemistry of this major neurodegenerative disease. In this paper, ten central, current challenges of the major paradigm in the field, the amyloid hypothesis, are sharply formulated. These challenges together show that new approaches are necessary that address data heterogeneity, increase focus on the proteome level, use available human patient data more actively, account for the aging phenotype as a background model of the disease, unify our understanding of the interplay between genetic and non-genetic risk factors, and combine into one framework both the familial and sporadic forms of the disease.
Alzheimer's Disease (AD) is a highly complex disease involving a broad range of clinical, cellular, and biochemical manifestations that are currently not understood in combination. This has led to many views of AD, e.g. the amyloid, tau, presenilin, oxidative stress, and metal hypotheses. The amyloid hypothesis has dominated the field with its assumption that buildup of pathogenic β-amyloid (Aβ) peptide causes disease. This paradigm has been criticized, yet most data suggest that Aβ plays a key role in the disease. Here, a new loss-of-function hypothesis is synthesized that accounts for the anomalies of the amyloid hypothesis, e.g. the curious pathogenicity of the Aβ42/Aβ40 ratio, the loss of Aβ caused by presenilin mutation, the mixed phenotypes of APP mutations, the poor clinical-biochemical correlations for genetic variant carriers, and the failure of Aβ reducing drugs. The amyloid-loss view accounts for recent findings on the structure and chemical features of Aβ variants and their coupling to human patient data. The lost normal function of APP/Aβ is argued to be metal transport across neuronal membranes, a view with no apparent anomalies and substantially more explanatory power than the gain-of-function amyloid hypothesis. In the loss-of-function scenario, the central event of Aβ aggregation is interpreted as a loss of soluble, functional monomer Aβ rather than toxic overload of oligomers. Accordingly, new research models and treatment strategies should focus on remediation of the functional amyloid balance, rather than strict containment of Aβ, which, for reasons rationalized in this review, has failed clinically.
In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta- amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1–38 or 25–35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.
Amylin is a 37-amino acid cytotoxic constituent of amyloid deposits found in the islets of Langerhans of patients with type
II diabetes. Extracellular accumulation of this peptide results in damage to insulin-producing β cell membranes and cell death.
We report here that at cytotoxic concentrations, amylin forms voltage-dependent, relatively nonselective, ion-permeable channels
in planar phospholipid bilayer membranes. Channel formation is dependent upon lipid membrane composition, ionic strength,
and membrane potential. At 1-10 μM, cytotoxic human amylin dramatically increases the conductance of lipid bilayer membranes,
while noncytotoxic rat amylin does not. We suggest that channel formation may be the mechanism of cytotoxicity of human amylin.
Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.
Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid β peptide (Aβ). Brain Aβ-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified Aβ-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that Aβ-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.
The amyloid beta protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid beta protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid beta protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid beta protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid beta protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid beta protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.
We have recently shown that the Alzheimer disease 40-residue amyloid beta-protein [A beta P-(1-40)] can form cation-selective channels when incorporated into planar lipid bilayers by fusion of liposomes containing the peptide. Since A beta P-(1-40) comprises portions of the putative extracellular and membrane-spanning domains of the amyloid precursor protein (APP751), we suggested that the channel-forming property could be the underlying cause of amyloid neurotoxicity. The peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, and we now report that soluble A beta P-(1-40) can also form similar channels in solvent-free lipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome-mediated incorporation, the amyloid channel activity in the patch pipet exhibits multiple conductance levels between 40 and 400 pS, cation selectivity, and sensitivity to tromethamine (Tris). Further studies with A beta P channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective. Amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would be homeostatically dissipative. We therefore interpret these data as providing further support for the concept that cell death in Alzheimer disease may be due to amyloid ion-channel activity.
The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca(2+)-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphate-mediated release of intracellular Ca2+.
The cellular mechanism underlying the generation of beta-amyloid in Alzheimer disease and its relationship to the normal metabolism of the amyloid precursor protein are unknown. In this report, we show that 3- and 4-kDa peptides derived from amyloid precursor protein are normally secreted. Epitope mapping and radiolabel sequence analysis suggest that the 4-kDa peptide is closely related to full-length beta-amyloid and the 3-kDa species is a heterogeneous set of peptides truncated at the beta-amyloid N terminus. The beta-amyloid peptides are secreted in parallel with amyloid precursor protein. Inhibitors of Golgi processing inhibit secretion of beta-amyloid peptides, whereas lysosomal inhibitors have no effect. The secretion of beta-amyloid-related peptides occurs in a wide variety of cell types, but which peptides are produced and their absolute levels are dependent on cell type. Human astrocytes generated higher levels of beta-amyloid than any other cell type examined. These results suggest that beta-amyloid is generated in the secretory pathway and provide evidence that glial cells are a major source of beta-amyloid production in the brain.
The Alzheimer disease 40-residue amyloid beta protein (AbetaP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AbetaP[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the AbetaP[1-40] channel. Provided the AbetaP[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose- dependent manner. For AbetaP[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates AbetaP[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on AbetaP[1-40] channels indicating asymmetric Zn(2+)-AbetaP[1-40] interactions at the entrance to the pore.
Amyloid β protein (AβP) forms senile plaques in the brain of the patients with Alzheimer’s disease. The early-onset AD has
been correlated with an increased level of 42-residue AβP (AβP1–42). However, very little is known about the role of AβP1–42 in such pathology. We have examined the activity of AβP1–42 reconstituted in phospholipid vesicles. Vesicles reconstituted with AβP show strong immunofluorescence labeling with an antibody
raised against an extracellular domain of AβP suggesting the incorporation of AβP peptide in the vesicular membrane. Vesicles
reconstituted with AβP showed a significant level of 45Ca2+ uptake. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of AβP, (ii) Tris, and (iii) Zn2+. However, reducing agents Trolox and dithiothreitol did not inhibit the 45Ca2+uptake, indicating that the oxidation of AβP or its surrounding lipid molecules is not directly involved in the AβP-mediated
Ca2+ uptake. An atomic force microscope was used to image the structure and physical properties of these vesicles. Vesicles ranged
from 0.5 to 1 μm in diameter. The stiffness of the AβP-containing vesicles was significantly higher in the presence of calcium.
The stiffness change was prevented in the presence of zinc, Tris, and anti-AβP antibody but not in the presence of Trolox
and dithiothreitol. Thus the stiffness change is consistent with the vesicular uptake of Ca2+. These findings provide biochemical and structural evidence that AβP1–42 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in Alzheimer’s
disease.
Studies on the amyloid precursor protein (APP) have suggested that it may be neuroprotective against amyloid-beta (Abeta) toxicity and oxidative stress. However, these findings have been obtained from either transfection of cell lines and mice that overexpress human APP isoforms or pretreatment of APP-expressing primary neurons with exogenous soluble APP. The neuroprotective role of endogenously expressed APP in neurons exposed to Abeta or oxidative stress has not been determined. This was investigated using primary cortical and cerebellar neuronal cultures established from APP knock-out (APP-/-) and wild-type (APP+/+) mice. Differences in susceptibility to Abeta toxicity or oxidative stress were not found between APP-/- and APP+/+ neurons. This observation may reflect the expression of the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) molecules and supports the theory that APP and the APLPs may have similar functional activities. Increased expression of cell-associated APLP2, but not APLP1, was detected in Abeta-treated APP-/- and APP+/+ cultures but not in H2O2-treated cultures. This suggests that the Abeta toxicity pathway differs from other general forms of oxidative stress. These findings show that Abeta toxicity does not require an interaction of the Abeta peptide with the parental molecule (APP) and is therefore distinct from prion protein neurotoxicity that is dependent on the expression of the parental cellular prion protein.
Stable transfectants of PC12 cells expressing bcl-2 or crmA were generated and tested for their susceptibility to various apoptotic insults. Bcl-2 expression conferred resistance to apoptosis induced by staurosporine and by oxidative insults including hydrogen peroxide and peroxynitrite, but was less effective in inhibition of activation-induced programmed cell death induced by concanavalin A. Concanavalin A-induced apoptosis was abated, however, in cells expressing very high levels of bcl-2. In contrast, cells expressing crmA were protected from concanavalin A-induced apoptosis, but were as susceptible as control cells to apoptosis induced by staurosporine and oxidative insults. Therefore, at least two apoptotic pathways in PC12 cells can be discerned by their differential sensitivity to blockade by bcl-2 and crmA. The ability of beta-amyloid (Abeta) to induce apoptosis in these cells was assessed. CrmA transfectants were protected from apoptosis induced by Abeta1-42, but only cells expressing very high levels of bcl-2 were similarly protected. These results suggest that the apoptotic pathway activated by Abeta1-42 in PC12 cells can be differentiated from the apoptotic pathway activated by oxidative insults. Gene transfer experiments also demonstrated that expression of crmA in primary cultures of hippocampal neurons is protective against cell death induced by Abeta1-42. Together these results support the hypothesis that Abeta-induced apoptosis occurs through activation-induced programmed cell death.
NIDDM is characterized by islet amyloid deposits and decreased beta-cell mass. Islet amyloid is derived from the locally expressed protein islet amyloid polypeptide (IAPP). While it is now widely accepted that abnormal aggregation of IAPP has a role in beta-cell death in NIDDM, the mechanism remains unknown. We hypothesized that small IAPP aggregates, rather than mature large amyloid deposits, are cytotoxic. Consistent with this hypothesis, freshly dissolved human (h)-IAPP was cytotoxic when added to dispersed mouse and human islet cells, provoking the formation of abnormal vesicle-like membrane structures in association with vacuolization and cell death. Human islet cell death occurred by both apoptosis and necrosis, predominantly between 24 and 48 h after exposure to h-IAPP. In contrast, the addition to dispersed islet cells of matured h-IAPP containing large amyloid deposits of organized fibrils was seldom associated with vesicle-like structures or features of cell death, even though the cells were often encased in the larger amyloid deposits. Based on these observations, we hypothesized that h-IAPP cytotoxicity is mediated by membrane damage induced by early h-IAPP aggregates. Consistent with this hypothesis, application of freshly dissolved h-IAPP to voltage-clamped planar bilayer membranes (a cell-free in vitro system) also caused membrane instability manifested as a marked increase in conductance, increased membrane electrical noise, and accelerated membrane breakage, effects that were absent using matured h-IAPP or rat IAPP solutions. Light-scattering techniques showed that membrane toxicity corresponded to h-IAPP aggregates containing approximately 25-6,000 IAPP molecules, an intermediate-sized amyloid particle that we term intermediate-sized toxic amyloid particles (ISTAPs). We conclude that freshly dissolved h-IAPP is cytotoxic and that this cytotoxicity is mediated through an interaction of ISTAPs with cellular membranes. Once ISTAPs mature into amyloid deposits comprising >10(6) molecules, the capacity of h-IAPP to cause membrane instability and islet cell death is significantly reduced or abolished. These data may have implications for the mechanism of cell death in other diseases characterized by local amyloid formation (such as Alzheimer's disease).
We examined vascular amyloid-beta deposition and other abnormalities in the posterior cerebral artery of consecutive cases of Alzheimer's disease (AD) compared to controls. Smooth muscle atrophy was a consistent feature in the cases of AD examined (p < 0.01) and was surprisingly independent of adjacent amyloid-beta deposition. These findings suggest that vascular abnormalities are a consistent feature in AD and may be an important contributor to the pathogenesis and complications of AD. (C) 1998 Elsevier Science B.V.
The beta-amyloid (A beta 1-40) peptide has previously been shown to enhance phenylephrine contraction of aortic rings in vitro. We have employed a novel observation, that A beta peptides enhance endothelin-1 (ET-1) contraction, to examine the relationship between vasoactivity and potential amyloidogenicity of A beta peptides, the role played by free radicals and calcium in the vasoactive mechanism, and the requirement of an intact endothelial layer for enhancement of vasoactivity. Rings of rat aortae were constricted with ET-1 before and after addition of amyloid peptide and/or other compounds, and a comparison was made between post- and pre-treatment contractions. In this system, vessel constriction is consistently dramatically enhanced by A beta 1-40, is enhanced less so by A beta 1-42, and is not enhanced by A beta 25-35. The endothelium is not required for A beta vasoactivity, and calcium channel blockers have a greater effect than antioxidants in blocking enhancement of vasoconstriction by A beta peptides. In contrast to A beta-induced cytotoxicity, A beta-induced vasoactivity is immediate, occurs in response to low doses of freshly solubilized peptide, and appears to be inversely related to the amyloidogenic potential of the A beta peptides. We conclude that the mechanism of A beta vasoactivity is distinct from that of A beta cytotoxicity. Although free radicals appear to modulate the vasoactive effects, the lack of requirement for endothelium suggests that loss of the free radical balance (between NO and O2-) may be a secondary influence on A beta enhancement of vasoconstriction. These effects of A beta on isolated vessels, and reported effects of A beta in cells of the vasculature, suggest that A beta-induced disruption of vascular tone may be a factor in the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease. Although the mechanism of enhanced vasoconstriction is unknown, it is reasonable to propose that in vivo contact of A beta peptides with small cerebral vessels may increase their tendency to constrict and oppose their tendency to relax. The subclinical ischemia resulting from this would be expected to up-regulate beta APP production in and around the vasculature with further increase in A beta formation and deposition. The disruptive and degenerative effects of such a cycle would lead to the complete destruction of cerebral vessels and consequently neuronal degeneration in the affected areas.
In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.
Glutamate neurotoxicity may be an underlying pathological mechanism contributing to neuronal cell loss in a variety of conditions including Alzheimer's disease (AD). In this study, we examined whether the beta-amyloid protein found in the neuritic plaques of AD alters the susceptibility of neurons to excitotoxic damage. While mature cortical neurons exposed to beta-amyloid protein for 2-4 days did not appear to be damaged, their vulnerability to low-intensity exposure to glutamate, N-methyl-D-aspartate, and kainate increased, suggesting that this mechanism may contribute to the neurodegeneration seen in AD.
The direct neurotoxic action of the beta-amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic-mediated neuronal death induced by beta-amyloid appears to reside in its ability to form Ca(2+)-permeable pores in neuronal membranes resulting in an excessive influx of Ca2+ and the induction of neurotoxic cascades. It is possible that during beta-amyloid exposure a Ca(2+)-mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of beta-amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to "aged" beta-amyloid25-35 peptide (5-50 microM) induced a slow, concentration-dependent apoptotic neurotoxicity (25-85%) during a 6 day exposure. Co-incubation of cultures with beta-amyloid25-35 peptide (25 microM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG-monomethyl-L-arginine [1 mM), N omega-nitro-L-arginine [1 mM], oxypurinol [100 microM], allopurinol [100 microM]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate beta-amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase-A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 microM), indomethacin (50 microM), and nor-dihydroguaiaretic acid (0.5 microM), respectively, did not reduce the proclivity of beta-amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 microM), vitamin C (100 microM), glutathione (100 microM), L-cysteine (100 microM), N-acetyl-cysteine (100 microM), deferoxamine (5 microM), or haemoglobin (35 micrograms/ml) failed to attenuate the neurotoxic action of beta-amyloid. On the other hand, pre-treatment of cultures with subtoxic concentrations of beta-amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that beta-amyloid peptide renders neurons more sensitive to free radical attack. However, a potential beta-amyloid-mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of beta-amyloid in vitro.
Despite the close morphological association of beta-amyloid and vascular cells, the functional effects of amyloid in cerebral endothelial cells in Alzheimer's disease have not been assessed. In this study, effects of amyloid fractions purified from senile plaques of AD brains were compared to synthetic amyloid peptides for their ability to affect brain endothelial cells in vitro. Our results indicate that plaque-derived amyloid inhibit brain endothelial cell proliferation in vitro by 40%. This inhibition was specific for plaque-derived amyloid, was not evoked by synthetic A beta 1-40, and was not mediated by alterations in intracellular calcium levels. Amyloid fractions from AD brains, although not directly toxic to brain endothelial cells, inhibit endothelial replication in vitro and therefore could alter the ability of vessels to repair and regenerate after injury.
An atomic force microscope was used to image the morphology and structural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from approximately 2-3 microns above the nucleus to approximately 20-30 nm over the distal processes, and cytoskeletal fibers as small as 30 nm wide were observed. Imaging over an extended period of time showed a limited degree of cytoskeletal reorganization. Localized force dissection did not induce significant retraction of cellular processes and immediate cell death. Differentiating PC-12 cells with a neuronal phenotype had a nonuniform morphology, abundant cytoskeletal elements, neuritic processes, and growth cones. The cell thickness varied from approximately 5-8 microns over the nucleus to approximately 100-500 nm over the neuritic processes; growth cones approximately 50-700 nm wide and end structures approximately 30-150 nm wide were visible. Repeated imaging showed reorganization of the growth cone, especially the appearance and disappearance of beadlike features and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.
Autoantibodies against glial fibrillary acidic protein (GFAP) and S100 protein were measured in sera of patients suffering from vascular dementia (VD), presenile Alzheimer's disease (AD), senile Alzheimer's disease (SDAT) and aged healthy controls by means of ELISA test. VD and SDAT showed the highest levels of both autoantibodies, AD the lowest. From these results a relationship between autoantibody titers and aging seems possible. Dosage of anti-GFAP and anti-S100 autoantibodies does not appear useful for diagnostic purpose because of the overlap observed among groups. Rather, the presence of these antibodies seems to reflect an alteration of the blood-brain barrier that promotes the access of central nervous system antigens to immunocompetent cells.
In cortical cultures, A beta protein destabilizes calcium homeostasis, but direct neurotoxicity of A beta is not observed. In hippocampal cultures, we and others find treatment with A beta protein decreases neuronal survival, but the mechanism of neurotoxicity is unknown. We have used low-density, serum-free cultures of hippocampal neurons to determine whether the neurotoxicity of A beta protein in vitro can be altered by voltage- or ligand-gated calcium channel antagonists or cyclic nucleotides. In these cultures, neither omega-conotoxin, nifedipine, verapamil, APV, nor MK-801 altered the survival of neurons exposed to synthetic A beta 1-40. The N-channel antagonist diltiazem decreased A beta 1-40 toxicity repeatedly, but slightly, perhaps by indirectly contributing to increased neuronal viability. Treatment of cultures with dibutyryl cAMP, 8-bromo cAMP, dibutyryl cGMP, and 8-bromo cGMP also failed to alter A beta toxicity. Thus, the toxicity of beta protein in low-density hippocampal cultures was not directly altered either by calcium channel blockers or by the addition of cyclic nucleotides.
The stresses acting on the luminal surface of endothelial cells due to shear flow were determined on a subcellular scale. Atomic force microscopy was used to measure the surface topography of confluent endothelial monolayers cultured under no-flow conditions or exposed to steady shear stress (12 dyn/cm2 for 24 h). Flow over these surface geometries was simulated by computational fluid dynamics, and the distribution of shear stress on the cell surface was calculated. Flow perturbations due to the undulating surface produced cell-scale variations of shear stress magnitude and hence large shear stress gradients. Reorganization of the endothelial surface in response to prolonged exposure to steady flow resulted in significant reductions in the peak shear stresses and shear stress gradients. From the relationship between surface geometry and the resulting shear stress distribution, we have defined a hydrodynamic shape factor that characterizes the three-dimensional morphological response of endothelial cells to flow. The analysis provides a complete description of the spatial distribution of stresses on individual endothelial cells within a confluent monolayer on a scale relevant to the study of physical mechanisms of mechanotransduction.
Several factors have highlighted the vasculature in Alzheimer's disease (AD): Cerebral amyloid angiopathy (CAA) is common, amyloid fibrils emanate from the vascular basement membrane (VBM), and similar forms of beta-amyloid are found in vascular and parenchymal amyloid accumulations. The present article discusses the presence of microvascular pathology in AD. Microangiopathy, in addition to neurofibrillary tangles, senile plaques, and CAA, is a common pathologic hallmark of AD. VBM components are associated with amyloid plaques, and nonamyloidotic alterations of the VBM occur in brain regions susceptible to AD lesions. Also, intra-VBM perivascular cells (traditionally called pericytes), a subset of which share the immunophenotype of microglia and other mononuclear phagocytic system (MPS) cells, have been implicated in vascular alterations and cerebrovascular amyloid deposition. Perivascular and parenchymal MPS cells have access to several sources of the beta-amyloid protein precursor, including platelets, circulating white cells, and neurons. MPS cells would thus be ideally situated to uptake and process the precursor, and deposit beta-amyloid in a fashion analogous to that seen in other forms of systemic and cerebral amyloidoses.
Alzheimer's disease (AD) and vascular dementia (VD) are the two most common causes of dementia. As yet, no definitive biological antemortem marker has been established for differential diagnosis of AD or VD. In this study, proteins of cerebrospinal fluid (CSF) from AD, VD and control patients were analyzed by two-dimensional (2-D) electrophoresis with immobilized pH gradients in the first dimension. No specific changes for AD or VD could be detected in the 2-D CSF patterns. However, a spot of haptoglobin alpha-1 chains (13.5 kDa; approximate pI 4.6) was found to be present in the majority of 2-D CSF maps from the dementia cases, suggesting a high-molecular-weight transudate type of alteration in the blood-brain barrier with considerable frequency in AD.
Alterations of the cerebral microvasculature have been reported in aging and in neurodegenerative disorders such as Alzheimer's disease. However, the exact role of microvascular alterations in the pathogenesis of neurodegeneration remains unknown. In the present report, the cerebral cortex microvasculature was studied by immunohistochemistry using a monoclonal antibody against vascular heparan sulfate proteoglycan protein core in normal aging controls. Alzheimer's disease, Down syndrome, Guam amyotrophic lateral sclerosis/parkinsonian dementia complex, Pick's disease and dementia pugilistica. In all dementing illnesses, increased microvascular pathology was evident compared to normal controls. Decreased microvascular density and numerous atrophic vessels were the primary abnormalities observed in all dementing disorders. These microvascular abnormalities demonstrated regional and laminar selectivity, and were primarily found in layers III and V of frontal and temporal cortex. Quantitative analysis employing computer-assisted microscopy demonstrated that the decrease in microvascular density in Alzheimer's disease was statistically significant compared to age-matched controls. In addition, extracellular heparan sulfate proteoglycan deposits were observed which colocalized with thioflavine S-positive senile plaques in Alzheimer's disease, Down syndrome and selected Guam dementia cases. In some cases, heparan sulfate proteoglycan was seen in senile plaques that appeared to be diffuse or primitive plaques that stained weakly with thioflavine. Heparan sulfate proteoglycan-containing neurons were also observed in Alzheimer's disease, as well as in Down syndrome and Guam cases. Glial staining for heparan sulfate proteoglycan was never observed. Our data support previous observations that microvascular pathology is found in aging and in Alzheimer's disease. The changes in Alzheimer's disease exceed those found in normal aging controls. We also found microvascular pathology in all other dementing disorders studied. Our studies further demonstrated that the microvascular pathology displays regional and laminar patterns which parallel patterns of neuronal loss. Finally, we also found that heparan sulfate proteoglycan is present in senile plaques and neurons not only as previously reported in Alzheimer's disease, but also in Down syndrome and Guam cases. Heparan sulfate proteoglycan in senile plaques may be derived from either the degenerating microvasculature or from degenerating neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
An in vitro tissue culture cell model system for investigating the biochemical mechanisms involved in the neurodegenerative actions of beta-amyloid has been established. Using rat pheochromocytoma PC12 cells, it was found that an early, specific response of cells to the beta-amyloid protein or the beta-amyloid fragment 25-35 was a potent inhibition of cellular redox activity, as measured by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction. This inhibitory response was rapid and occurred at nanomolar concentrations of peptide, concentrations at which no equivalent decreases in cell proliferation or cell survival were observed. The inhibition of PC12 cell MTT reduction was initially reversible upon removal of the peptide; if sustained for several days, however, by repeated peptide application, it became associated with a dramatic reduction in cell survival. Inhibition of MTT reduction may, therefore, be an early indicator of the mechanism of beta-amyloid-mediated cell death.
Although beta-amyloid is the main constituent of neurite plaques and may play a role in the pathophysiology of Alzheimer's
disease, mechanisms by which soluble beta-amyloid might produce early symptoms such as memory loss before diffuse plaque deposition
have not been implicated. Treatment of fibroblasts with beta-amyloid (10 nM) induced the same potassium channel dysfunction
previously shown to occur specifically in fibroblasts from patients with Alzheimer's disease--namely, the absence of a 113-picosiemen
potassium channel. A tetraethylammonium-induced increase of intracellular concentrations of calcium, [Ca2+]i, a response that
depends on functional 113-picosiemen potassium channels, was also eliminated or markedly reduced by 10 nM beta-amyloid. Increased
[Ca2+]i induced by high concentrations of extracellular potassium and 166-picosiemen potassium channels were unaffected by
10 nM beta-amyloid. In Alzheimer's disease, then, beta-amyloid might alter potassium channels and thus impair neuronal function
to produce symptoms such as memory loss by a means other than plaque formation.
Deposition of beta/A4 amyloid in brain is a defining characteristic of Alzheimer disease (AD); however, the extent to which amyloid deposits may interfere with normal cellular processes is incompletely understood. We examined this issue by means of PC12 cells. After transfection with DNA coding for 97 amino acids of the beta/A4 C-terminal region of the amyloid precursor protein, beta/A4 antigen was visible at the cell membrane. We report that normal unstimulated PC12 cells exhibit ruffling activity at the cell surface when plated on a plastic substrate. Relative to control cells, however, those that over-expressed the beta/A4 C-terminal peptide had significantly higher levels of ruffling activity, suggesting a structural and/or functional membrane modification. Similar cellular alterations, if present, in Alzheimer brain cells, may indicate disturbances in membrane-associated functions, including intercellular communication.
The newly developed atomic force microscope (AFM) provides a unique window to the microworld of cells, subcellular structures, and biomolecules. The AFM can image the three-dimensional structure of biological specimens in a physiological environment. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. The process of image acquisition is such that the AFM can also measure forces at the molecular level. In addition, the AFM can interact with the sample, thereby manipulating the molecules in a defined manner--nanomanipulation! The AFM has been used to image living cells and the underlying cytoskeleton, chromatin and plasmids, ion channels, and a variety of membranes. Dynamic processes such as crystal growth and the polymerization of fibrinogen and physicochemical properties such as elasticity and viscosity in living cells have been studied. Nanomanipulations, including dissection of DNA, plasma membranes, and cells, and transfer of synthetic structures have been achieved. This review describes the operating principles, accomplishments, and the future promise of the AFM.
Alzheimer's disease has a multifactorial pathogenesis. Among the various factors involved, this review examines, in particular, the possibility of oxidative stress, meaning an imbalance between the formation and spread of reactive oxygen species (ROS) and the antioxidant defenses. This theory is supported by the following observations: (a) the alteration of mitochondrial function, which is likely to lead to the electron leakage in the respiratory chain and the consequent formation of superoxide radicals; (b) the unbalanced high activity of superoxide dismutase and monoamine oxidase B which causes the production of more H2O2; (c) the alteration of iron homeostasis which, in combination with the superoxide and H2O2, gives rise to the most deleterious hydroxyl radicals; (d) the increased lipid peroxidation and membrane alterations; (e) the pro-aggregating effect of ROS on beta/A4 protein and the C-terminal fragment of amyloid precursor (A4CT). Most of these changes are already present in the normal aging brain but are aggravated in AD presumably over a number of years. However, further investigations are needed to confirm these theories particularly regarding the alterations of another target of ROS, the proteins. Peroxidative stress is presumably present in the AD brain. This stress might not be a primary factor in the pathogenesis of AD, but a consequence of the tissue injury. In any case, it could contribute considerably to the pathology, in a vicious cycle of actions and reactions resulting in a critical mass of metabolic errors, responsible in the end for this disease.
Deposits of beta-amyloid are apparent in ageing and Alzheimer's disease, but the role of this peptide in neurodegeneration is unclear. The free-radical theory of ageing may also account for Alzheimer-type degeneration and consequently links between free-radical generation and beta-amyloid have been sought. We demonstrate here that beta-amyloid interacts with endothelial cells on blood vessels to produce and excess of superoxide radicals, with attendant alterations in endothelial structure and function. The superoxide radical can scavenge endothelium-derived relaxing factor and produce potent oxidizing agents, which can cause lipid peroxidation and other degenerative changes. The alterations in vascular tone and endothelial damage are prevented by the oxygen-radical-scavenging enzyme superoxide dismutase. These observations suggest a normal vasoactive role for beta-amyloid as well as a mechanism by which beta-amyloid may play a role in vascular abnormalities and neurodegeneration mediated by free radicals.
To determine whether the presenilin 1 (PS1), presenilin 2 (PS2) and amyloid beta-protein precursor (APP) mutations linked to familial Alzheimer's disease (FAD) increase the extracellular concentration of amyloid beta-protein (A beta) ending at A beta 42(43) in vivo, we performed a blinded comparison of plasma A beta levels in carriers of these mutations and controls. A beta 1-42(43) was elevated in plasma from subjects with FAD-linked PS1 (P < 0.0001), PS2N1411 (P = 0.009), APPK670N,M671L (P < 0.0001), and APPV7171 (one subject) mutations. A beta ending at A beta 42(43) was also significantly elevated in fibroblast media from subjects with PS1 (P < 0.0001) or PS2 (P = 0.03) mutations. These findings indicate that the FAD-linked mutations may all cause Alzhelmer's disease by increasing the extracellular concentration of A beta 42(43), thereby fostering cerebral deposition of this highly amyloidogenic peptide.
Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.
Mutations in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early-onset, autosomal dominant Alzheimer's disease. Studies of plasma and fibroblasts from subjects with these mutations have established that they all alter amyloid beta-protein (beta APP) processing, which normally leads to the secretion of amyloid-beta protein (relative molecular mass 4,000; M(r) 4K; approximately 90% A beta1-40, approximately 10% A beta1-42(43)), so that the extracellular concentration of A beta42(43) is increased. This increase in A beta42(43) is believed to be the critical change that initiates Alzheimer's disease pathogenesis because A beta42(43) is deposited early and selectively in the senile plaques that are observed in the brains of patients with all forms of the disease. To establish that the presenilin mutations increase the amount of A beta42(43) in the brain and to test whether presenilin mutations act as true (gain of function) dominants, we have now constructed mice expressing wild-type and mutant presenilin genes. Analysis of these mice showed that overexpression of mutant, but not wild-type, PS1 selectively increases brain A beta42(43). These results indicate that the presenilin mutations probably cause Alzheimer's disease through a gain of deleterious function that increases the amount of A beta42(43) in the brain.
Prions cause neurodegenerative disease in animals and humans. Recently it was shown that a 21-residue fragment of the prion protein (106-126) could be toxic to cultured neurons. We report here that this peptide forms ion-permeable channels in planar lipid bilayer membranes. These channels are freely permeable to common physiological ions, and their formation is significantly enhanced by "aging" and/or low pH. We suggest that channel formation is the cytotoxic mechanism of action of amyloidogenic peptides found in prion-related encephalopathies and other amyloidoses. The channels reported here are large enough and nonselective enough to mediate cell death through discharge of cellular membrane potential, changes in ionic homeostasis, and specifically, influx of calcium, perhaps triggering apoptosis.
We report that human hNT cells display neuron-like calcium channel activation. Patch-clamp experiments show that exposure of hNT cells to the Alzheimer-related amyloid peptide beta AP(25-35) induces large and irreversible inward calcium currents at -80 mV in whole cell mode, with a linear current-voltage relationship. This behavior is suggestive of ionophore formation. An analogous peptide with scrambled sequence has no effect. These ionophore effects by the beta AP(25-35) peptide, the first report in a human cell-line, are very rapid effects. The currents are large and stable, and are blocked by Al3+ but not by Cd2+. Filtration removes a peptide aggregate from the amyloid peptide beta AP(25-35) solution and thereby abolishes the inward current. The residual soluble peptide has no effect. These data suggest that the initial step of the neurotoxic effect of beta AP(25-35) may be due to the insertion of the aggregated peptide into the cellular membrane as a Ca2(+)-carrying ionophore. The relevance of calcium-mediated cell death, especially in Alzheimer's disease, is discussed.
Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.
Amyloid beta-peptide (A beta) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that A beta25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to A beta25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by A beta25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of A beta25-35 were specific because A beta1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of A beta1-40 aggregates and because astrocytes did not undergo similar changes after exposure to A beta25-35. Damage and death of ECs induced by A beta25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that A beta induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then A beta and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.
Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic alpha-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Deltah5, 6) mutant did not. The most frequent conductance observed (18 +/- 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 +/- 2 pS and 90 +/- 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.
We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.
In an attempt to elucidate the relationship among aggregation properties, fiber morphology, and cellular toxicity several β-amyloid peptides (Aβ) were prepared according to a standardized procedure. Peptides either carried mutations inside the membrane anchor segment around amino acid position 35 or their carboxy terminus was shortened from 42 to 41, 40, or 39 amino acids. The time-dependent self-assembly of monomeric Aβ into fibers was simultaneously monitored by electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, and Aβ-mediated cellular toxicity using the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. The transition of Aβ monomers into fibers was analyzed by more than 600 electron micrographs. Distinct morphological changes from seed-like structures to immature and mature fibers were observed. Seeds were of spherical appearance. Immature fibers were typically elongated structures with a rough surface and with varying thickness depending on the Aβ sequence. Mature fibers were characterized by a periodic variation of their thickness along the fiber axis. The proportion of these different structures and the total amount of aggregated Aβ was amino acid sequence-dependent. Wild-type Aβ1-42and its oxidized derivative carrying a methionine sulfoxide residue at position 35 showed the highest rate of fiber formation and exerted toxic activity in the MTT assay at very low nanomolar concentrations. The fibers formed by these two peptides were predominantly of the mature type. In contrast, carboxyl-terminus truncated peptides Aβ1-41, Aβ1-40, and Aβ1-39or most Aβ1-42derivatives mutated around amino acid position 35 showed a reduced aggregation rate, the immature fibers predominated, and the toxicity was orders of magnitude lower. Thus, a correlation can be drawn among the chemical structure, aggregation properties, fiber morphology, and cellular toxicity.
Most cases of early-onset familial Alzheimer's disease (FAD) are caused by mutations in the genes encoding the presenilin
1 (PS1) and PS2 proteins, both of which undergo regulated endoproteolytic processing. During apoptosis, PS1 and PS2 were shown
to be cleaved at sites distal to their normal cleavage sites by a caspase-3 family protease. In cells expressing PS2 containing
the asparagine-141 FAD mutant, the ratio of alternative to normal PS2 cleavage fragments was increased relative to wild-type
PS2-expressing cells, suggesting a potential role for apoptosis-associated cleavage of presenilins in the pathogenesis of
Alzheimer's disease.
Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis)
either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to
form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and
acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release
at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic
effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.
The 39-42 amino acid residue amyloid beta peptide (A beta), the major protein component in senile plaques and cerebrovascular amyloidosis in the brain in Alzheimer's disease (AD), has been shown to be neurotoxic in vitro. Accumulating data from several areas suggest that cerebrovascular dysfunction and damage may also play a significant role in the AD process. For instance, we have recently demonstrated enhanced vasoconstriction and resistance to relaxation in intact rat aorta treated with A beta [Thomas et al., beta-Amyloid-mediated vasoactivity and vascular endothelial damage, Nature, 380 (1996) 168-171]. Significant vessel damage occurred after thirty minutes of exposure, but could be prevented with superoxide dismutase. To further investigate the role of A beta toxicity on endothelial cells, we have applied A beta peptides to cultures of human aortic endothelial cells (HAEC). Our results show that both A beta(1-42) and A beta(25-35) are toxic to HAEC in a time- and dose-dependent manner, and that this toxicity can be partially prevented by the calcium channel blocker, verapamil, and the antioxidant, superoxide dismutase. The common form of A beta, A beta(1-40), which has been shown to be neurotoxic, is much less toxic to HAEC. A beta toxicity to HAEC occurs within 30 min of treatment with relatively lower doses than those usually observed in primary cultured neurons and vascular smooth muscle cells. It was recently reported that a variety of mutations in the beta-amyloid protein precursor gene and the Presenilin-1 and -2 genes linked to early-onset familial AD cause an increase in the plasma concentration of A beta(1-42) in mutation carriers [Scheuner et al., Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vitro by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease, Nature Med., 2 (1996) 864-870]. Human aortic endothelial cells are more sensitive to A beta(1-42) than A beta(1-40), via a pathway involving an excess of superoxide free radicals and influx of extracellular calcium. Finally, we have evidence that both apoptotic and necrotic processes are activated by the A beta peptides in these endothelial cells.
Mutations in two related genes, presenilin 1 and 2, account for most early-onset familial Alzheimer's disease. Although structural features indicate that the presenilins are membrane proteins, their function(s) is unknown. We have localized the presenilins to the nuclear membrane, its associated interphase kinetochores, and the centrosomes-all subcellular structures involved in cell cycle regulation and mitosis. The colocalization of the presenilins with kinetochores on the nucleoplasmic surface of the inner nuclear membrane, together with other results, suggests that they may play a role in chromosome organization and segregation, perhaps as kinetochore binding proteins/receptors. We discuss a pathogenic pathway for familial Alzheimer's disease in which defective presenilin function causes chromosome missegregation during mitosis, resulting in apoptosis and/or trisomy 21 mosaicism.
beta-Amyloid precursor protein (beta-APP), the source of the fibrillogenic amyloid beta-peptide (A beta) that accumulates in the brain of victims of Alzheimer's disease, is a multifunctional protein that is widely expressed in the nervous system. beta-Amyloid precursor protein is axonally transported and accumulates in presynaptic terminals and growth cones. A secreted form of beta-APP (sAPP alpha) is released from neurons in response to electrical activity and may function in modulation of neuronal excitability, synaptic plasticity, neurite outgrowth, synaptogenesis, and cell survival. A signaling pathway involving guanosine 3',5'-cyclic monophosphate is activated by sAPP alpha and modulates the activities of potassium channels, N-methyl-D-aspartate receptors, and the transcription factor NF kappa B. Additional functions of beta-APP may include modulation of cell adhesion and regulation of proliferation of nonneuronal cells. Alternative enzymatic processing of beta-APP liberates A beta, which has a propensity to form amyloid fibrils; A beta can damage and kill neurons and increase their vulnerability to excitotoxicity. The mechanism involves generation of oxyradicals and impairment of membrane transport systems (e.g., ion-motive ATPases and glutamate and glucose transporters). Genetic mutations or age-related metabolic changes may promote neuronal degeneration in Alzheimer's disease by increasing production of A beta and/or decreasing levels of neuroprotective sAPP alpha.
The formation of fibrillar deposits of amyloid beta protein (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). A central question is whether Abeta plays a direct role in the neurodegenerative process in AD. The involvement of Abeta in the neurodegenerative process is suggested by the neurotoxicity of the fibrillar form of Abeta in vitro. However, mice transgenic for the Abeta precursor protein that develop amyloid deposits in the brain do not show the degree of neuronal loss or tau phosphorylation found in AD. Here we show that microinjection of plaque-equivalent concentrations of fibrillar, but not soluble, Abeta in the aged rhesus monkey cerebral cortex results in profound neuronal loss, tau phosphorylation and microglial proliferation. Fibrillar Abeta at plaque-equivalent concentrations is not toxic in the young adult rhesus brain. Abeta toxicity in vivo is also highly species-specific; toxicity is greater in aged rhesus monkeys than in aged marmoset monkeys, and is not significant in aged rats. These results suggest that Abeta neurotoxicity in vivo is a pathological response of the aging brain, which is most pronounced in higher order primates. Thus, longevity may contribute to the unique susceptibility of humans to Alzheimer's disease by rendering the brain vulnerable to Abeta neurotoxicity.
Perhaps the most reproducible early event induced by the interaction of amyloid beta peptide (A beta) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as A beta and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with beta-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by A beta. NAC35, an amyloidogenic fragment of alpha-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the beta-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with beta-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute A beta neurotoxicity.