Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression. Special issue dedicated to Dr. Moussa Youdim.     

A Comparative Study of β-Amyloid Peptides Aβ1-42 and Aβ25-35 Toxicity in Organotypic Hippocampal Slice Cultures

Accumulation of the neurotoxic amyloid β-peptide (Aβ) in the brain is a hallmark of Alzheimer’s disease (AD). Several synthetic Aβ peptides have been used to study the mechanisms of toxicity. Here, we sought to establish comparability between two commonly used Aβ peptides Aβ1-42 and Aβ25-35 on an in vitro model of Aβ toxicity. For this purpose we used organotypic slice cultures of rat hippocampus and observed that both Aβ peptides caused similar toxic effects regarding to propidium iodide uptake and caspase-3 activation. In addition, we also did not observe any effect of both peptides on Akt and PTEN phosphorylation; otherwise the phosphorylation of GSK-3β was increased. Although further studies are necessary for understanding mechanisms underlying Aβ peptide toxicity, our results provide strong evidence that Aβ1-42 and the Aβ25-35 peptides induce neural injury in a similar pattern and that Aβ25-35 is a convenient tool for the investigation of neurotoxic mechanisms involved in AD.     

Cholesterol Potentiates β-Amyloid-Induced Toxicity in Human Neuroblastoma Cells: Involvement of Oxidative Stress

Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer’s disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid β peptide (Aβ) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Aβ peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Aβ, show a significant increase in membrane-associated oxidative stress. Since Aβ is able to promote oxidative stress directly by catalytically producing H₂O₂ from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Aβ-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin. Special issue article in honor of Dr. Ricardo Tapia.     

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.     

Molecular modeling of zinc and copper binding with Alzheimer’s amyloid β-peptide

Aggregation of the amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer’s disease. Cu(II) and Zn(II) ions were reported to be able to induce Aβ aggregation at nearly physiological concentrations in vitro. In this study, the binding modes of Cu(II) and Zn(II) in this process were explored by molecular modeling. In the pre-associated Aβ, Nτ atom of imidazole ring of His14, O atom of carbonyl of main-chain and two O atoms of water occupied the four ligand positions of the complex. While in the aggregated form of Aβ, the His13(N)–Metals–His14(N) bridges were formed through metal cross-linking action. These results would be helpful to put insight on revealing the formation mechanism of pathogenic Aβ aggregates in brain.     

Metal effects on the membrane interactions of amyloid-β peptides

Aβ(1–42) peptide, found as aggregated species in Alzheimer’s disease brain, is linked to the onset of dementia. We detail results of ³¹P and ²H solid-state NMR studies of model membranes with Aβ peptides and the effect of metal ions (Cu²⁺ and Zn²⁺), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when Aβ(1–42) is associated with the bilayer membrane. Aβ(25–35), a fragment from the C-terminal end of Aβ(1–42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with Aβ(25–35) peptides. ³¹P magic angle spinning NMR data show that Aβ(1–42) and Aβ(1–42)-Cu²⁺ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state ¹³C NMR was used to compare structural changes of Aβ(1–42) to those of Aβ(25–35) in model membrane systems of anionic phospholipids and cholesterol. The Aβ peptides appeared to have an increase in β-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu²⁺ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated Aβ/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Neprilysin Deficiency-Dependent Impairment of Cognitive Functions in a Mouse Model of Amyloidosis

Alzheimer’s disease, responsible for the vast majority of dementia cases in the elderly population, is caused by accumulation of toxic levels of amyloid β peptide (Aβ) in the brain. Neprilysin is a major enzyme responsible for the degradation of Aβ in vivo. We have previously shown that elevation of neprilysin levels in the brain delays the deposition of Aβ -plaques in a mouse model of amyloidosis and that lack of neprilysin leads to increased Aβ generation and to signs of incipient neurodegeneration in mouse brains. This study was designed to test whether low brain levels of neprilysin affect the amyloid pathology or perturb the learning and memory performance of mice. Double-mutated mice carrying a targeted depletion of one allele of Mme, the gene encoding neprilysin, and over-expressing human amyloid precursor protein (APP), exhibited a reinforced amyloid pathology in comparison with their APP transgenic littermates. Moreover, in contrast to their parental lines, these mice were impaired in the Morris water maze learning and memory paradigm and showed facilitated extinction in the conditioned taste aversion test. These data suggest that even a partial neprilysin deficiency, as is found during aging, exacerbates amyloid pathology and may impair cognitive functions. Special issue to Honor Dr. Akitane Mori.     

The molecular genetics of Alzheimer's disease

The major pathological characteristic of Alzheimer's disease (AD) is the abnormal deposition of β-amyloid peptide (Aβ) in the brain. In some early onset cases, the disease develops because of mutations in the gene coding for β-amyloid precursor protein (βAPP). However, the majority of AD families in the early onset subgroup are linked to a locus on chromosome 14. The genetic analysis and age of onset correlates of both the βAPP gene and the chromosome 14 locus are discussed. We speculate on the mechanisms by which the βAPP mutations cause the disease and discuss recent advances in βAPP processing that may be relevant to the pathogenesis of the late-onset (common) form of the disease. In addition, we review the association of theAPOE locus with late-onset familial and nonfamilial disease. Further work is required to establish the effects of this locus on disease occurrence, age of onset, and progression. The molecular pathology of ApoE in relation to AD development and the identification of the chromosome 14 gene will greatly contribute to a general pathogenic model of AD, and will clarify the role of βAPP and its derivatives.     

Alzheimer’s Disease: Halothane Induces Aβ Peptide to Oligomeric Form—Solution NMR Studies

Alzheimer’s disease (AD) is a significant contributor to cognitive decline and is responsible for about half of the cases of dementia in later life. Although exact etiology of AD is not known, however, many risk factors for AD are identified. Anesthesia for elderly patients is considered as a risk factor in AD as they frequently experience deterioration in cognitive function with long exposure to anesthetics during surgery. Inhaled anesthetic agents remain the mainstay for patients undergoing major surgical operations. This study using multidimensional NMR spectroscopy provides the first direct evidence in vitro that inhaled anesthetic, halothane specifically interacts with Aβ40 and Aβ42 peptide. Halothane induces structural alternation of Aβ peptide from soluble monomeric α-helical form to oligomeric β-sheet conformation, which may hasten the onset of AD. Aβ42 is more prone to oligomerization compared to Aβ40 in the presence of halothane. The molecular mechanism of halothane induced structural alternation of Aβ peptide is discussed. An erratum to this article can be found at     

Cholesterol inhibits the insertion of the Alzheimer’s peptide Aβ(25–35) in lipid bilayers

The physiological relationship between brain cholesterol content and the action of amyloid β (Aβ) peptide in Alzheimer’s disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Aβ/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Aβ(25–35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Aβ(25–35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Aβ(25–35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Aβ(25–35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties.     

Secondary structural modifications of Aβ(1-40) induced by multiple 2-acetoxy-4-methoxybenzyl (acetylHmb) protection

Summary Modifications to secondary structure and fibril formation caused by multiple acetylHmb backbone amide protection of Alzheimer's disease Aβ(1–40) were investigated using circular dichroism spectroscopy and electron microscopy. Penta(acetylHmb) Aβ(1–40) was observed to have a reduced ability to form α-helix and β-sheet structures under the same solution conditions as the native peptide, with α-helical propensity being reduced more significantly than β-sheet propensity. Further, acetylHmb backbone protection was found to alter Aβ(1–40) interaction with SDS-micelles by preventing α-helix formation. Aβ fibril formation, a characteristic property of this peptide, was also not observed for penta(acetylHmb) Aβ(1–40).     

Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells

Background  

Self-assembly of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting Aβ self-assembly could serve as therapeutic agents and endogenous molecules that modulate Aβ self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Aβ aggregates warns that inhibition at intermediate stages of Aβ self-assembly may prove detrimental. Here, we explore the inhibition of Aβ_1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Aβ_1–40 activation of endothelial cells for monocyte adhesion.     

Computational study of the binding of CuII to Alzheimer’s amyloid-β peptide: Do Aβ42 and Aβ40 bind copper in identical fashion?

One of the many hypotheses on the pathogenesis of Alzheimer’s disease is that the amyloid-β peptide (Aβ) binds Cu^II and can catalytically generate H₂O₂, leading to oxidative damage in brain tissues. For a molecular level understanding of such catalysis it is critical to know the structure of the Aβ–Cu^II complex precisely. Unfortunately, no high-resolution structure is available to date and there is considerable debate over the copper coordination environment with no clear consensus on which residues are directly bound to Cu^II. Considering all plausible isomers of the copper-bound Aβ42 and Aβ40 using a combination of density functional theory and classical molecular dynamics methods, we report an atomic resolution structure for each possible complex. We evaluated the relative energies of these isomeric structures and surprisingly found that Aβ42 and Aβ40 display very different binding modes, suggesting that shorter peptides that are truncated at the C-terminus may not be realistic models for understanding the chemistry of the most neurotoxic peptide, Aβ42. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and use of human brain microvascular endothelial cells to examine β-amyloid exchange in the blood-brain barrier

Alzheimer’s disease (AD) is characterized by excessive cerebrovascular deposition of the β-amyloid peptide (Aβ). The investigation of Aβ transport across the blood-brain barrier (BBB) has been hindered by inherent limitations in the cellular systems currently used to model the BBB, such as insufficient barrier properties and poor reproducibility. In addition, many of the existing models are not of human or brain origin and are often arduous to establish and maintain. Thus, we characterized an in vitro model of the BBB employing human brain microvascular endothelial cells (HBMEC) and evaluated its utility to investigate Aβ exchange at the blood-brain interface. Our HBMEC model offers an ease of culture compared with primary isolated or coculture BBB models and is more representative of the human brain endothelium than many of the cell lines currently used to study the BBB. In our studies, the HBMEC model exhibited barrier properties comparable to existing BBB models as evidenced by the restricted permeability of a known paracellular marker. In addition, using a simple and rapid fluormetric assay, we showed that antagonism of key Aβ transport proteins significantly altered the bi-directional transcytosis of fluorescein-Aβ (1–42) across the HBMEC model. Moreover, the magnitude of these effects was consistent with reports in the literature using the same ligands in existing in vitro models of the BBB. These studies establish the HBMEC as a representative in vitro model of the BBB and offer a rapid fluorometric method of assessing Aβ exchange between the periphery and the brain.     

Vitamin D, tamoxifen and β-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium

Reciprocal interactions between tumor cells and endothelial cells constitute the most important stage of tumor metastasis. There is growing evidence suggesting that β-estradiol and vitamin D modulate the progression of steroid-sensitive breast cancers. In keeping with those results, the purpose of the study reported here was to determine the cytotoxic and antiproliferative activity of tamoxifen (TAM) in the T47D human breast cancer cell line depending on the cell culture model (three-dimensional (3D, spheroid) or two-dimensional (2D, monolayer)) and to estimate the antiproliferative activity of vitamin D in balanced TAM/β-estradiol conditions. The study was also designed to investigate whether vitamin D might influence interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) production in a co-culture of T47D cell spheroids with an endothelial cell monolayer in the presence of β-estradiol and TAM. Spectrophotometric analysis with MTT revealed that the cytotoxic and antiproliferative activity of TAM was dependent on the culture model, the density of cell culture, and culture medium supplements. In balanced TAM/β-estradiol medium, vitamin D only slightly inhibited T47D cell proliferation in both 2D and 3D cultures. Direct contact of tumor cell spheroids with the endothelium induced production of MMP-2 and IL-6, which was significantly inhibited in TAM/β-estradiol balanced medium. Addition of vitamin D further inhibited MMP-2 production, but enhanced the production of IL-6 as was shown by ELISA assay. Our co-culture model in TAM/β-estradiol balanced medium proved to be useful for examining direct and paracrine interactions of tumor cells with the endothelium in conditions that were closer to in vivo conditions than in the standard 2D model.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.     

Neuroprotectin D1 Induces Neuronal Survival and Downregulation of Amyloidogenic Processing in Alzheimer’s Disease Cellular Models

The mediator neuroprotectin D1 (NPD1) is an enzymatic derivative of the omega-3 essential fatty acid docosahexaenoic acid. NPD1 stereoselectively and specifically binds to human retinal pigment epithelium (RPE) cells and neutrophils. In turn, this lipid mediator induces dephosphorylation of Bcl-x_L in a PP2A-dependent manner and induces PI3K/Akt and mTOR/p70S6K pathways leading to RPE cell survival during oxidative stress-induced apoptosis. As a proof of principle of its systemic in vivo bioactivity, NPD1 attenuates laser-induced choroidal neovascularization in mice. Using human neural cells transfected with amyloid precursor protein (APP)sw (Swedish double mutation APP695sw, K595N, M596L), NPD1 was shown to regulate secretase-mediated production of Aβ peptide, downregulates pro-inflammatory gene expression, and promotes cell survival. In human neural cells overexpressing beta-amyloid precursor protein (βAPP), the lipid mediator suppressed Aβ42 shedding by downregulating β-secretase (BACE1) while activating the α-secretase (ADAM10), thus shifting the βAPP cleavage from the noxious amyloidogenic pathway into a non-amyloidogenic, neurotrophic pathway. Furthermore, downregulation of Aβ42 peptide release by NPD1 may be dependent upon PPARγ activation. In conclusion, NPD1 exhibits anti-inflammatory, anti-amyloidogenic, and anti-apoptotic bioactivities in human neural cells in part via PPARγ signaling and through the targeting of α- and β-secretase systems.     

Gene transfer of a hybrid interleukin-1β gene to B16 mouse melanoma recruits leucocyte subsets and reduces tumour growth in vivo

 Interleukin(IL)-1 differs from most other cytokines in its lack of a signal sequence. This results in intracellular retention of the immature proform. The release of IL-1 has been shown to be restricted predominantly to activated monocytes and macrophages and to be associated with apoptosis of the producer cell. These features have limited the investigation of IL-1 in early immune responses. In order to study the biological effects of local IL-1β release during an antitumour immune response, we used B16 mouse melanoma cells transduced with mature human IL-1β cDNA constructs. To obtain a released form of human IL-1β (ssIL-1β), the signal sequence from the related IL-1 receptor antagonist was ligated to the cDNA that encoded the mature form of IL-1β. When cells of the poorly immunogenic B16 melanoma cell line were transduced with IL-1β by retroviral infection, high levels of the protein were detected intracellularly, whereas cells transduced with IL-1β containing the signal sequence secreted most of their protein. The in vitro growth of the melanoma cells was unaffected by the IL-1β or ssIL-1β gene transfer. In contrast, the in vivo subcutaneous tumour growth of the ssIL-1β-transduced B16 cells in syngeneic C57BL/6 mice was significantly reduced compared with the IL-1β- and the mock-transduced controls. Immunohistochemical analysis revealed the infiltration of macrophages to be strong in B16/ssIL-1β, moderate in B16/IL-1β and minimal in control tumours. Furthermore, a moderate infiltration of CD4⁺ cells and of scattered dendritic cells was detected in B16/ssIL-1β tumours whereas very few or no CD4⁺ cells and dendritic cells were seen in the B16/IL-1β or control tumours. Following in vivo growth, all the tumours up-regulated ICAM-1 on their cell surfaces. However, the percentage of ICAM-1-expressing cells was two- to fourfold higher in B16/ssIL-1β tumours compared to the control. The data suggest that IL-1β acts in vivo, either directly or indirectly, as a chemotactic factor for monocytes, T helper cells and dendritic cells. This supports IL-1β having a regulatory effect on tumour growth when locally released in the tumour area. Received: 12 November 1996 / Accepted: 6 May 1997