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.     

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.     

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     

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.     

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.     

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.     

X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

γ‐Secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aβ

Deposition of β ‐amyloid (Aβ) peptides, cleavage products of β‐amyloid precursor protein (APP) by β‐secretase‐1 (BACE1) and γ‐secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ‐Secretase inhibition is a therapeutical anti‐Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti‐Aβ efficacy. The present study compared active γ‐secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [³H]‐L‐685,458, a radiolabeled high‐affinity γ‐secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post‐mortem delays. The CP in post‐mortem samples exhibited exceptionally high [³H]‐L‐685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin‐1 immunoreactivity, and β‐site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ‐secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non‐neuronal contributor to CSF Aβ, probably at reduced levels in AD.     

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.     

Acyl ghrelin improves cognition,synaptic plasticity deficits and neuroinflammation following amyloid β (Aβ1‐40) administration in mice

Ghrelin is a metabolic hormone that has neuroprotective actions in a number of neurological conditions, including Parkinson's disease (PD), stroke and traumatic brain injury. Acyl ghrelin treatment in vivo and in vitro also shows protective capacity in Alzheimer's disease (AD). In the present study, we used ghrelin knockout (KO) and their wild‐type littermates to test whether or not endogenous ghrelin is protective in a mouse model of AD, in which human amyloid β peptide 1‐40 (Aβ_1‐40) was injected into the lateral ventricles i.c.v. Recognition memory, using the novel object recognition task, was significantly impaired in ghrelin KO mice and after i.c.v. Aβ_1‐40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Spatial orientation, as assessed by the Y‐maze task, was also significantly impaired in ghrelin KO mice and after i.c.v. Aβ_1‐40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Ghrelin KO mice had deficits in olfactory discrimination; however, neither i.c.v. Aβ_1‐40 treatment, nor acyl ghrelin injections affected olfactory discrimination. We used stereology to show that ghrelin KO and Aβ_1‐40 increased the total number of glial fibrillary acidic protein expressing astrocytes and ionised calcium‐binding adapter expressing microglial in the rostral hippocampus. Finally, Aβ_1‐40 blocked long‐term potentiation induced by high‐frequency stimulation and this effect could be acutely blocked with co‐administration of acyl ghrelin. Collectively, our studies demonstrate that ghrelin deletion affects memory performance and also that acyl ghrelin treatment may delay the onset of early events of AD. This supports the idea that acyl ghrelin treatment may be therapeutically beneficial with respect to restricting disease progression in AD.     

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.     

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.