Astrocytic Aβ1‐42 uptake is determined by Aβ‐aggregation state and the presence of amyloid‐associated proteins

Intracerebral accumulation of amyloid‐β (Aβ) leading to Aβ plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective Aβ‐clearance. We previously found primary human astrocytes and microglia able to bind and ingest Aβ1‐42 in vitro, which appeared to be limited by Aβ1‐42 fibril formation. We now confirm that astrocytic Aβ‐uptake depends on size and/or composition of Aβ‐aggregates as astrocytes preferably take up oligomeric Aβ over fibrillar Aβ. Upon exposure to either fluorescence‐labelled Aβ1‐42 oligomers (Aβ_oligo) or fibrils (Aβ_fib), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Aβ‐internalization was verified using confocal microscopy and live‐cell imaging. Neither uptake of Aβ_oligo nor Aβ_fib, triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin‐6 and monocyte‐chemoattractant protein‐1 release. Amyloid‐associated proteins, including α1‐antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Aβ‐aggregation. Here, astrocytic uptake of Aβ_fib increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Aβ_oligo‐positive astrocytes, whereas SAP/C1q slightly reduced Aβ_oligo uptake. Thus, amyloid‐associated proteins, especially ApoJ and ApoE, can alter Aβ‐uptake in vitro and hence may influence Aβ clearance and plaque formation in vivo. © 2010 Wiley‐Liss, Inc.     

Characterization of plasma β‐secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease

Reduction in cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and elevation in total tau and phospho‐thr181 tau consistently differentiate between Alzheimer's disease (AD) and age‐matched control subjects. In contrast, CSF β‐site APP‐cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and β (sAPPα and sAPPβ) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPβ, and sAPPα assays and investigated their ability to distinguish AD from age‐matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1‐specific inhibitor. Plasma sAPPβ and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ‐secretase inhibitor led to significant lowering of plasma sAPPβ with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPβ, sAPPα, and Aβ42 (P < 0.001) compared with age‐matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies. © 2012 Wiley Periodicals, 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.     

Caspases‐2 and ‐8 are involved in the presenilin1/γ‐secretase‐dependent cleavage of amyloid precursor protein after the induction of apoptosis

The presenilin/γ‐secretase protease cleaves many type‐I membrane proteins, including the amyloid β‐protein (Aβ) precursor (APP). Previous studies have shown that apoptosis induces alterations in Aβ production in a caspase‐dependent manner. Here, we report that staurosporine (STS)‐induced apoptosis induces caspase‐8 and/or‐2‐dependent γ‐secretase activation. Blocking of caspase activity with caspase‐8 inhibitor z‐IETD‐fmk, and caspase‐2 inhibitor z‐VDVAD‐fmk reduced Aβ production by STS in H4 cells expressing the Swedish mutant of APP (HSW) or APP‐C99 (H4‐C99). There was no inhibitory effect of other caspases (‐1, ‐3, ‐5, ‐6, ‐9) on Aβ production by STS. This finding was further supported by evidence that siRNA transfection, depleting caspase‐2 or ‐8 levels, lowered Aβ production in HSW and H4‐C99 cells without affecting expression of APP or γ‐secretase complex. In addition, Aβ production by STS was decreased by JNK inhibitors, SP600125. These results suggest that caspase‐2 and/or ‐8 is involved in presenilin/γ‐secretase activation and Aβ production in apoptosis. © 2010 Wiley‐Liss, Inc.     

Presenilin 1 is involved in the maturation of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)

One of the pathologic hallmarks of Alzheimer's disease is the excessive deposition of beta-amyloid peptides (Abeta) in senile plaques. Abeta is generated when beta-amyloid precursor protein (APP) is cleaved sequentially by beta-secretase, identified as beta-site APP-cleaving enzyme 1 (BACE1), and gamma-secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of Abeta peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin-deficient mouse embryonic fibroblasts (PS-/-MEFs) were reduced significantly compared to those in wild-type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH-SY5Y, stably expressing wild-type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant-negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH-SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both beta- and gamma-secretase in Abeta generation.     

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.     

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.     

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.     

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.     

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques in the brain. Amyloid-beta peptide (Abeta) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Abeta generation. In support of this, APP, Abeta, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Abeta are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT-1 binds to BACE1 (beta-site APP cleaving enzyme 1) and that overexpression of CAV-1 or FLOT-1 results in recruiting BACE1 into lipid rafts and influence on beta-secretase activity in cultured cells. Our results show that both CAV-1 and FLOT-1 may modulate beta-secretase activity by interacting with BACE1.     

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.     

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.     

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.