Secreted amyloid precursor protein and holo‐APP bind amyloid β through distinct domains eliciting different toxic responses on hippocampal neurons

Amyloid β (Aβ) is a metabolic product of Aβ precursor protein (APP). Deposition of Aβ in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Aβ induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor‐like protein for Aβ fibrils (fAβ). In this study, we present further experimental support for the direct interaction of APP with fAβ and for its involvement in Aβ neurotoxicity. Using recombinant purified holo‐APP (h‐APP), we have shown that it directly binds fAβ. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAβ. One sequence in the n‐terminus of APP is required for binding of fAβ to secreted APP (s‐APP) but not to h‐APP. In addition, the extracellular juxtamembrane Aβ‐sequence mediates binding of fAβ to h‐APP but not to s‐APP. Deletion of the extracellular juxtamembrane Aβ sequence abolishes abnormal h‐APP accumulation and toxicity induced by fAβ deposition, whereas deletions in the n‐terminus of APP do not affect Aβ toxicity. These experiments show that interaction of toxic Aβ species with its membrane‐anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Aβ‐receptor‐like function of APP directly implicated in neuronal degeneration in AD. © 2010 Wiley‐Liss, Inc.     

Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABA_A modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABA_A antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABA_A receptors. © 2013 Wiley Periodicals, Inc.     

The E693Δ (Osaka) mutation in amyloid precursor protein potentiates cholesterol‐mediated intracellular amyloid β toxicity via its impaired cholesterol efflux

It has been shown that amyloid β (Aβ) secretion regulates cholesterol efflux from cells and that the E693Δ (Osaka) mutation in amyloid precursor protein (APP) promotes intracellular accumulation of Aβ and thus reduces its secretion. These findings led us to speculate that APP with the Osaka mutation (APP_OSK) might have a defect in cholesterol efflux and thus cause cellular malfunction. We therefore examined the effects of this mutation on intracellular cholesterol transport and efflux in cultured cells. Upon cholesterol loading, APP_OSK‐expressing cells exhibited higher levels of cellular cholesterol than wild‐type APP‐expressing cells, suggesting impaired cholesterol efflux. It is known that, after its internalization, cholesterol is transported from the endosomes to the endoplasmic reticulum (ER) and Golgi apparatus and then to the plasma membrane. In APP_OSK‐expressing cells, cholesterol accumulated with Aβ in the ER and Golgi apparatus and alone in endosomes/lysosomes. These results imply that the mutation‐induced disturbance of Aβ trafficking from the ER to the plasma membrane affects cholesterol transport to cause cholesterol accumulation in the ER and Golgi apparatus and, consequently, in endosomes. Furthermore, we detected an enhanced mitochondrial accumulation of Aβ and cholesterol in APP_OSK‐expressing cells, and this was accompanied by an increase in the generation of reactive oxygen species (ROS). The present findings suggest that Aβ trafficking is important for intracellular cholesterol transport and efflux and that the Osaka mutation potentiates cholesterol‐dependent exacerbation of intracellular Aβ toxicity, i.e. Aβ‐induced ROS generation, by disturbing Aβ‐mediated cholesterol efflux from the cell. © 2013 Wiley Periodicals, Inc.     

Characteristics of cerebral β amyloid deposition in four non-demented patients in their forties with a high apolipoprotein E ε4 allele frequency

Four autopsied brains from mentally normal patients aged 43–49 who had cerebral β amyloid deposition were examined. Three patients had breast cancer, and in one of these cases it was associated with brain metastasis and brain radiation therapy. One other case had pulmonary small cell carcinoma. In two patients, small β amyloid deposits were only found in the frontal cortex. In another two patients, β amyloid deposits were found in many cortical areas and the density of plaques was higher than in the former two patients. No β amyloid deposition was found in the cerebella, basal ganglia, or brain stem in any of the four patients. When examined with end-specific antisera for the C-terminal of amyloid β protein (Aβ), Aβ42 was predominant in the diffuse plaques and immunoreactions for Aβ40 varied among the patients. The N-terminal of Aβ was truncated in a subset of plaques. Tau- and phosphorylated tau-reactive fine neurites were only found in the entorhinal cortex of Case 3. The apoE genotype of these four patients were 3/4, 3/4, 4/4 and 3/3, and therefore, the ε4 allele frequency (50%) was as high as that in AD. Three out of four patients had at least one ε4 allele, a risk factor of AD. There is a possibility that these subjects might develop AD when in their seventies. It may take 30 years from the beginning of Aβ deposition to the clinical manifestation of dementia.     

NMDA receptor–dependent signaling pathways that underlie amyloid β‐protein disruption of LTP in the hippocampus

Alzheimer's disease (AD), the most common neurodegenerative disease in the elderly population, is characterized by the hippocampal deposition of fibrils formed by amyloid β‐protein (Aβ), a 40‐ to 42‐amino‐acid peptide. The folding of Aβ into neurotoxic oligomeric, protofibrillar, and fibrillar assemblies is believed to mediate the key pathologic event in AD. The hippocampus is especially susceptible in AD and early degenerative symptoms include significant deficits in the performance of hippocampal‐dependent cognitive abilities such as spatial learning and memory. Transgenic mouse models of AD that express C‐terminal segments or mutant variants of amyloid precursor protein, the protein from which Aβ is derived, exhibit age‐dependent spatial memory impairment and attenuated long‐term potentiation (LTP) in the hippocampal CA1 and dentate gyrus (DG) regions. Recent experimental evidence suggests that Aβ disturbs N‐methyl‐D ‐aspartic acid (NMDA) receptor–dependent LTP induction in the CA1 and DG both in vivo and in vitro. Furthermore, these studies suggest that Aβ specifically interferes with several major signaling pathways downstream of the NMDA receptor, including the Ca²⁺‐dependent protein phosphatase calcineurin, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), protein phosphatase 1, and cAMP response element–binding protein (CREB). The influence of Aβ on each of these downstream effectors of the NMDA receptor is reviewed in this article. Additionally, other mechanisms of LTP modulation, such as Aβ attenuation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor currents, are briefly discussed. © 2009 Wiley‐Liss, Inc.     

Enhanced aggregation and β structure of amyloid β peptide after coincubation with C1Q

Several lines of evidence now suggest that aggregation of soluble amyloid β peptide (Aβ) into a cross β sheet configuration may be an important factor in mediating potential neurotoxicity of Aβ. Synthetic Aβ has been shown to self aggregate in vitro. Here, we demonstrate that coincubation of freshly solubilized Aβ with C1q, a complement component known to bind Aβ in vitro and to colocalize with Aβ in vivo, results in as much as a 7-fold enhancement of Aβ aggregation, as well as a 2–4-fold enhancement of β structure within aggregates. The addition of C1q to preformed Aβ aggregates also results in significantly increased resistance to aggregate resolubilization. © 1994 Wiley-Liss, Inc.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Relative efficacies of amyloid β peptide (Aβ) binding proteins in Aβ aggregation

The aggregation of amyloid β peptide (Aβ) into its fibrillar, cross β-pleated configuration is generally viewed as a critical event in the pathophysiology of Alzheimer's disease (AD). A diverse group of molecules, the Aβ binding proteins, has been evaluated for their effects on this process. However, most of these studies have used micromolar or greater reagent concentrations, and their different methods have not permitted quantitative comparisons of the efficacy of different Aβ binding proteins in augmenting or inhibiting aggregation. In the present work we have undertaken a coherent analysis using fluorimetry of thioflavin T-stained experimental solutions. The complement protein C1q, serum amyloid P, and transthyretin significantly enhanced the formation of precipitable, cross β-pleated aggregates in solutions of 800 nM Aβ1–42. Under these same experimental conditions, α₁-antichymotrypsin had no significant effect on the aggregation process, and both the E3 and E4 isoforms of apolipoprotein E were significant inhibitors. There was a non-significant trend toward the E3 isoform exhibiting greater inhibition than the E4 isoform. Of the aggregation-facilitating molecules, C1q was substantially and significantly the most potent. © 1996 Wiley-Liss, Inc.     

β‐amyloid and ATP‐induced diffusional trapping of astrocyte and neuronal metabotropic glutamate type‐5 receptors

β‐Amyloid (Aβ) oligomers initiate synaptotoxicity following their interaction with the plasma membrane. Several proteins including metabotropic glutamate type 5 receptors (mGluR5s) contribute to this process. We observed an overexpression of mGluR5s in reactive astrocytes surrounding Aβ plaques in brain sections from an Alzheimer's disease mouse model. In a simplified cell culture system, using immunocytochemistry and single molecule imaging, we demonstrated a rapid binding of Aβ oligomers on the plasma membrane of astrocytes. The resulting aggregates of Aβ oligomers led to the diffusional trapping and clustering of mGluR5s. Further, Aβ oligomers induced an increase in ATP release following activation of astroglial mGluR5s by its agonist. ATP slowed mGluR5s diffusion in astrocytes as well as in neurons co‐cultured with astrocytes. This effect, which is purinergic receptor‐dependent, was not observed in pure neuronal cultures. Thus, Aβ oligomer‐ and mGluR5‐dependent ATP release by astrocytes may contribute to the overall deleterious effect of mGluR5s in Alzheimer's disease. GLIA 2013;61:1673–1686     

Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis

R. A. Armstrong and N. J. Cairns (2010) Neuropathology and Applied Neurobiology 36, 248–257
Analysis of β‐amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis Aim: To determine the spatial pattern of β‐amyloid (Aβ) deposition throughout the temporal lobe in Alzheimer's disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aβ. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aβ deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components were present with density peaks of Aβ deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits. Conclusions: (i) Aβ deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aβ deposition may reflect the formation of Aβ deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aβ deposition both in AD and in transgenic models of disease.     

Dual pathways mediate β‐amyloid stimulated glutathione release from astrocytes

Oxidative stress plays an important role in the progression of Alzheimer's disease (AD) and other neurodegenerative conditions. Glutathione (GSH), the major antioxidant in the central nervous system, is primarily synthesized and released by astrocytes. We determined if β‐amyloid (Aβ42), crucially involved in Alzheimer's disease, affected GSH release. Monomeric Aβ (mAβ) stimulated GSH release from cultured cortical astrocytes more effectively than oligomeric Aβ (oAβ) or fibrillary Aβ (fAβ). Monomeric Aβ increased the expression of the transporter ABCC1 (also referred to as MRP1) that is the main pathway for GSH release. GSH release from astrocytes, with or without mAβ stimulation, was reduced by pharmacological inhibition of ABCC1. Astrocytes robustly express connexin proteins, especially connexin43 (Cx43), and mAβ also stimulated Cx43 hemichannel‐mediated glutamate and GSH release. Aβ‐stimulation facilitated hemichannel opening in the presence of normal extracellular calcium by reducing astrocyte cholesterol level. Aβ treatment did not alter the intracellular concentration of reduced or oxidized glutathione. Using a mouse model of AD with early onset Aβ deposition (5xFAD), we found that cortical ABCC1 was significantly increased in temporal register with the surge of Aβ levels in these mice. ABCC1 levels remained elevated from 1.5 to 3.5 months of age in 5xFAD mice, before plunging to subcontrol levels when amyloid plaques appeared. Similarly, in cultured astrocytes, prolonged incubation with aggregated Aβ, but not mAβ, reduced induction of ABCC1 expression. These results support the hypothesis that in the early stage of AD pathogenesis, less aggregated Aβ increases GSH release from astrocytes (via ABCC1 transporters and Cx43 hemichannels) providing temporary protection from oxidative stress which promotes AD development. GLIA 2015;63:2208–2219     

The peripheral messenger RNA expression of glycogen synthase kinase‐3β genes in Alzheimer's disease patients: a preliminary study

Objective: To explore the peripheral leucocytic messenger RNA (mRNA) expression of glycogen synthase kinase‐3β (GSK‐3β) gene in Alzheimer's disease (AD) patients. Methods: Using TaqMan relative quantitative real‐time polymerase chain reaction, we analyzed leucocytic gene expression of GSK‐3β in 48 AD patients and 49 healthy controls. Clinical data of AD patients were also collected. Results: The mRNA expression level of the GSK‐3β gene was significantly higher in the AD group (3.13 ± 0.62) than in the normal group (2.77 ± 0.77). Correlational analyses showed that the mRNA expression level of GSK‐3β gene in AD patients was associated with the age of onset (P= 0.047), age (P= 0.055), and Behavioral Pathology in Alzheimer's Disease Rating Scale total score (P= 0.062) and subscores: aggressiveness score (P= 0.073) and anxieties and phobias score (P= 0.067). Through multivariate regression model, older age, higher anxieties and phobias score and aggressiveness score were associated with higher mRNA expression level of GSK‐3β gene. Conclusion: In AD patients, the mRNA expression level of the GSK‐3β gene is increased and may be related to age and behavioural pathology in AD.