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.     

Macrophage colony stimulating factor is associated with excretion of amyloid‐β peptides from cerebrospinal fluid to peripheral blood

Background: The process of aggregation of brain amyloid‐β peptides (Aβ) is thought to be associated with the pathogenesis of Alzheimer's disease (AD). Amyloid‐β peptides are produced by sequential endoproteolysis by β‐site amyloid‐β protein precursor‐cleaving enzyme (BACE) followed by presenilin (PS)/γ‐secretase. There are several species of Aβ due to cleavage diversity of PS/γ‐secretase. The predominant species in human cerebrospinal fluid (CSF) or plasma is Aβ40, whereas Aβ42 is much more aggregatable and accumulated in senile plaques. The level of Aβ in the brain is determined by the balance between the generation and clearance of Aβ, including transport across the brain–blood barrier (BBB). Although the processes of Aβ generation and degradation have been studied in some detail, knowledge of the Aβ transport process across the BBB is limited. So far, low‐density lipoprotein receptor‐related protein (LRP1), P‐glycoprotein (P‐gp), and insulin‐like growth factor‐1 (IGF‐1) have been identified to modify the excretion of brain Aβ to the blood. Methods: To investigate whether macrophage colony stimulating factor (M‐CSF) has a role in the Aβ transport process, human Aβ was injected into the lateral ventricle of the brain of M‐CSF‐deficient (op/op) mice. Then, plasma and brain Aβ levels were measured by ELISA to determine the time‐course of Aβ movement from the brain to the plasma. Result: When human Aβ40 was injected into mouse lateral ventricles, the efflux of Aβ from the CSF to the blood was transiently decreased and delayed in M‐CSF‐deficient mice. Moreover, endogenous plasma Aβ40 levels were lower in M‐CSF‐deficient mice. Conclusion: The results indicate that M‐CSF deficiency impairs excretion of human‐type Aβ40 from the CSF to blood. We propose that M‐CSF may be a novel factor that facilitates the excretion of Aβ from the CSF to the blood via the BBB.     

The two‐hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of β‐secretase BACE1

β‐Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a membrane‐bound protease that is essential for the production of β‐amyloid protein (Aβ). Given the crucial role of Aβ accumulation in Alzheimer's disease (AD), inhibition of BACE1 activity may represent a feasible therapeutic strategy in the treatment of AD. Recently, we and others identified reticulon 3 (RTN3) and reticulon 4‐B/C (RTN4‐B/C or Nogo‐B/C) as membrane proteins that interact with BACE1 and inhibit its ability to produce Aβ. In this study, we employed various mutants of RTN3 and RTN4‐C and C. elegans RTN to investigate the molecular mechanisms by which RTNs regulate BACE1. We found that RTN3 mutants lacking the N‐terminal or C‐terminal or loop domain as well as a RTN4‐C mutant lacking the C‐terminal domain bound to BACE1 comparably to wild‐type RTN3 and RTN4‐C. Furthermore, overexpression of wild‐type RTN3, RTN4‐C, and these RTN mutants similarly reduced Aβ40 and Aβ42 secretion by cells expressing Swedish mutant APP. C. elegans RTN, which has low homology to human RTNs, also interacted with BACE1 and inhibited Aβ secretion. In contrast, two RTN3 mutants containing deletions of the first or second potential transmembrane domains and an RTN3 swap mutant of the second transmembrane domain bound BACE1 but failed to inhibit Aβ secretion. Collectively, these results suggest that the two‐transmembrane‐domain tertiary structure of RTN proteins is critical for the ability of RTNs to modulate BACE1 activity, whereas N‐terminal, C‐terminal and loop regions are not essential for this function. © 2009 Wiley‐Liss, Inc.     

Conversion of Aβ43 to Aβ40 by the successive action of angiotensin‐converting enzyme 2 and angiotensin‐converting enzyme

The longer and neurotoxic species of amyloid‐β protein (Aβ), Aβ42 and Aβ43, contribute to Aβ accumulation in Alzheimer's disease (AD) pathogenesis and are considered to be the primary cause of the disease. In contrast, the predominant secreted form of Aβ, Aβ40, inhibits amyloid deposition and may have neuroprotective effects. We have reported that angiotensin‐converting enzyme (ACE) converts Aβ42 to Aβ40 and that Aβ43 is the earliest‐depositing Aβ species in the amyloid precursor protein transgenic mouse brain. Here we found that Aβ43 can be converted to Aβ42 and to Aβ40 in mouse brain lysate. We further identified the brain Aβ43‐to‐Aβ42‐converting enzyme as ACE2. The purified human ACE2 converted Aβ43 to Aβ42, and this activity was inhibited by a specific ACE2 inhibitor, DX600. Notably, the combination of ACE2 and ACE could convert Aβ43 to Aβ40. Our results indicate that the longer, neurotoxic forms of Aβ can be converted to the shorter, less toxic or neuroprotective forms of Aβ by ACE2 and ACE. Moreover, we found that ACE2 activity showed a tendency to decrease in the serum of AD patients compared with normal controls, suggesting an association between lower ACE2 activity and AD. Thus, maintaining brain ACE2 and ACE activities may be important for preventing brain amyloid neurotoxicity and deposition in Alzheimer's disease. © 2014 Wiley Periodicals, Inc.     

Cyclin‐Dependent kinase 5 targeting prevents β‐Amyloid aggregation involving glycogen synthase kinase 3β and phosphatases

Inappropriate activation of cyclin‐dependent kinase 5 (CDK5) resulting from proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles, β‐amyloid (βA) aggregation, and chronic neurodegeneration. At 18 months of age, 3× Tg‐AD mice were sacrificed after either 3 weeks (short term) or 1 year (long term) of CDK5 knockdown. In short‐term‐treated animals, CDK5 knockdown reversed βA aggregation in the hippocampi via inhibitory phosphorylation of glycogen synthase kinase 3β Ser9 and activation of phosphatase PP2A. In long‐term‐treated animals, CDK5 knockdown induced a persistent reduction in CDK5 and prevented βA aggregation, but the effect on amyloid precursor protein processing was reduced, suggesting that yearly booster therapy would be required. These findings further validate CDK5 as a target for preventing or blocking amyloidosis in older transgenic mice. © 2015 Wiley Periodicals, 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.     

2‐Phenylethynyl‐butyltellurium attenuates amyloid‐β peptide(25–35)‐induced learning and memory impairments in mice

Our previous study demonstrated that 2‐phenylethynyl‐butyltellurium (PEBT), an organotellurium compound, enhances memory in mice. In this study, the effects of PEBT on cognitive impairment induced by Aβ_25–35 were assessed by Morris water maze and step‐down inhibitory avoidance tasks. Mice received a single intracerebroventricular injection of Aβ_25–35 (3 nmol/3 μl/per site) and a daily oral administration of PEBT (1 mg/kg, for 10 days). PEBT significantly improved Aβ‐induced learning deficits on the training session in the Morris water maze. At the probe trial session, PEBT significantly decreased the escape latency and increased the number of crossings in the platform local compared with the Aβ‐treated group. PEBT significantly improved Aβ‐induced memory impairment in the step‐down inhibitory avoidance task. General locomotor activity was similar in all groups. This study showed that PEBT ameliorated the impairments of spatial and nonspatial long‐term memory evaluated on Morris water maze and step‐down inhibitory avoidance tasks, respectively. The results suggest that PEBT could be considered a candidate for the prevention of memory deficits such as those observed in Alzheimer's disease. © 2013 Wiley Periodicals, Inc.     

Alterations in spontaneous delta and gamma activity might provide clues to detect changes induced by amyloid‐β administration

Alzheimer's disease (AD) is the most prevalent form of dementia and has an increasing incidence. The neuropathogenesis of AD is suggested to be a result of the accumulation of amyloid‐β (Aβ) peptides in the brain. To date, Aβ‐induced cognitive and neurophysiologic impairments have not been illuminated sufficiently. Therefore, we aimed to examine how spontaneous brain activities of rats changed by injection of increasing Aβ doses into the brain hemispheres, and whether these changes could be used as a new biomarker for the early diagnosis of the AD. Rats were randomized into following groups: sham (Sham) and seven Aβ‐treated (i.c.v.) groups in increasing concentrations (from Aβ‐1 to Aβ‐7). After recovery, EEG recordings were obtained from implanted electrodes from eight electrode locations, and then, spectral and statistical analyses were performed. A significant decrement in gamma activity was observed in all Aβ groups compared with the sham group. In delta activity, we observed significant changes from Aβ‐4 to Aβ‐7 group compared with sham group. Delta coherence values were decreased from Aβ‐4 to Aβ‐7 and Aβ‐5 to Aβ‐7 groups for frontal and temporal electrode pairs, respectively. A gradual increment was observed in Aβ_1‐42 level till Aβ‐4 group. Positive correlation for global delta power and negative correlation for global gamma power between Aβ_1‐42 peptide levels were detected. Consequently, it is conceivable to suggest gamma oscillation might be used to detect early stages of AD. Moreover, changes in delta activity provide information about the onset of major pathologic changes in the progress of AD.     

Methyl 3,4‐dihydroxybenzoate protects primary cortical neurons against Aβ25–35‐induced neurotoxicity through mitochondria pathway

Amyloid‐β peptides (Aβ), which can aggregate into oligomers or fibrils in neurons, play a critical role in the pathogenesis of Alzheimer's disease (AD). Methyl 3,4‐dihydroxybenzoate (MDHB), a phenolic acid compound, has been reported to have antioxidative and neurotrophic effects. The present study investigated the neuroprotective effects of MDHB against Aβ‐induced apoptosis in rat primary cortical neutons. The primary cortical neurons were pretreated with different concentrations of MDHB for 24 hr, then incubated with 10 μM Aβ_25–35 for 24 hr. The results showed that Aβ_25–35 could induce neurotoxicity as evidenced by the decreased cell viability and the increased apoptotic rate. In parallel, Aβ_25–35 significantly increased the reactive oxygen species accumulation and decreased mitochondrial membrane potential. However, pretreatment of the primary cortical neurons with MDHB could effectively suppress these cellular events caused by Aβ_25–35 exposure. In addition, MDHB could increase the level of Bcl‐2, decrease the level of Bax, and inhibit the activation of caspase‐9 and caspase‐3 in Aβ_25–35‐treated primary cortical neurons. All these results were beneficial in their protective effect against Aβ‐induced neurotoxicity. Our results suggest that MDHB has a neuroprotective effect that provides a pharmacological basis for its clinical use in the treatment of AD. © 2013 Wiley Periodicals, Inc.     

Intracellular sAPP retention in response to Aβ is mapped to cytoskeleton‐associated structures

Amyloid β (Aβ) contributes to neurodegeneration in Alzheimer's disease and provides a close association between molecular events and pathology, although the underlying molecular mechanisms are unclear. In the work described here, Aβ did not induce amyloid precursor protein (APP) expression, but APP processing/trafficking was markedly affected. In COS‐7 cells, Aβ provokes retention of intracellular sAPPα (isAPPα). Intracellular holo‐APP levels remain unchanged, and extracellular total sAPP increases, although extracellular sAPPα alone was not altered significantly. In primary neuronal cultures and PC12 cells, isAPP also increased, but this was mirrored by a decrease in extracellular total sAPP. The isAPP retention was particularly associated with the cytoskeletal fraction. The retention “per se” occurred in vesicular‐like densities, negative for a C‐terminal antibody and strongly positive for the 6E10 antibody, clearly showing abnormal intracellular accumulation of sAPPα in response to Aβ. Our data support a dynamic model for intracellular retention of sAPPα as an early response to Aβ exposure. Particularly noteworthy was the observation that removal of Aβ reversed the isAPP accumulation. Mechanistically, these findings disclose an attractive physiological response, revealing the capacity of cells to deal with adverse effects induced by Aβ. © 2008 Wiley‐Liss, Inc.     

Increased frequency of the α‐1‐antichymotrypsin T allele in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a process of unknown etiology characterized by amyloid deposition in the wall of small cerebral and meningeal blood vessels. CAA is also a feature of Alzheimer's disease (AD) and of a subgroup of elderly people. α‐1‐Antichymotrypsin (ACT) is a serum glycoprotein frequently associated with vascular and senile plaque amyloid. The ACT gene is known to have a bi‐allele polymorphism that causes a simple amino acid substitution. In an attempt to clarify the possible role of ACT polymorphism in AD and in cases of CAA, the ACT genotype was investigated in AD, CAA, and intellectually intact controls. Representative brain areas (cerebral cortex, hippocampus, putamen, white matter, and gyrus cinguli) from all cases were studied using classical histologic staining techniques (hematoxylin–eosin (HE), Mallory's thrichromic or alkaline congo red stain), and immunohistochemistry for tau and β‐amyloid proteins. There was a significantly increased T allele and TT genotype frequency in the CAA group, but not in the AD group, suggesting a role for the ACT genotype in the development of vascular lesions. The presence of the apolipoprotein E4 allele (ApoE4) did not correlate with the ACT‐A allele, as previously reported, and appeared to be independent of the risk for developing AD.     

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background: During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42. Methods: Cell‐free γ‐secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI‐TOF MS analysis was carried out to detect the N‐termini of AICD species. Aβ and AICD species were measured by ELISA and immunoblotting techniques. Results: Effects on the ε‐cleavage by AD‐associated pathological mutations around the ε‐cleavage sites (i.e., βAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε‐cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aβ42 production. Only AICDε51, though, and not Aβ42 production, dramatically changed with modifications to the cell‐free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. Conclusion: Our current data show that the generation ratio of AICDε51 and Aβ42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aβ42 generation, measurement of the relative level of Aβ42‐related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε‐cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε‐cleavage as a means for decreasing Aβ42 production.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Active clearance of human amyloid β 1–42 peptide aggregates from the rat ventricular system

A major constituent of SP in the brains of Alzheimer's disease is 39–43 amino acid peptide called β‐amyloid peptide (Aβ). Recent data have demonstrated that Aβ has a strong tendency to form insoluble aggregates and that toxic effects of Aβ is based on its aggregation. In the current study, 100 µg of human synthetic Aβ 1–42 (sAβ 1–42) was infused into the lateral ventricle of rat brain using a short‐term infusion model. At 2 or 7 days following the infusion, sAβ 1–42 was found to form insoluble aggregates, scattering throughout the entire ventricular systems. The sAβ 1–42 aggregates were partially engulfed by phagocytic cells and deposited at the meningeal vessels or the choroid plexuses. However, these deposits mostly disappeared from the ventricles by 28 days post‐infusion. Here, it is reported for the first time that considerable amounts of sAβ 1–42 are almost cleared from the rat ventricular system by the mononuclear phagocytic system.