BACE1 Levels Research Articles

Hydrogen sulfide down-regulates BACE1 and PS1 via activating PI3K/Akt pathway in the brain of APP/PS1 transgenic mouse.

Endogenous hydrogen sulfide (H2S) may have multiple physiological functions in brain. Our previous study showed that H2S improved spatial memory impairment and decreased the production of Aβ in APP/PS1 transgenic mice. However, many of the underlying mechanisms are not still being elucidated. The aim of the present study is to investigate the neuroprotective mechanisms of H2S involving in the activity of β-secretase (BACE1), γ-secretase (PS1) and α-secretase (ADAM17). Morris water maze was used to measure the behavior change. The levels of Aβ40 and Aβ42 were quantified using colorimetric ELISA kits and immunohistochemical analysis. The levels of BACE1, PS1, ADAM17, pAkt, pp38MAPK, pERK and pJNK were tested by Western blot analysis in normal mice, APP/PS1 transgenic mice and 50μmol/kg-NaHS-treated transgenic mice. On the basis of exogenous H2S treatment, LY294002 (inhibitors of PI3K/Akt) or PD98059 (inhibitors of MAPK/ERK) was injected into lateral cerebral ventricle. The levels of BACE1, PS1 and pp38MAPK were increased and ADAM17 were decreased in the APP/PS1 transgenic mice. After intraperitoneal administration of an H2S donor (NaHS) into APP/PS1 mice, the levels of BACE1, PS1 and pp38MAPK were reduced and ADAM17 increased. The level of pp38 MAPKs, pAkt and pERK1/2 was increased in APP/PS1 transgenic mice compared with normal mice (p<0.05). There was no difference in the expression of pJNK between AD transgenic mice and normal mice (p>0.05). These results demonstrated that LY294002 inhibited the effect of H2S on decreasing the BACE1 and PS1, reducing the level of Aβ and improving memory impairment in APP/PS1 transgenic mice. PD98059 had no influence on the expression of BACE1 and PS1. H2S inhibits the expression of BACE1 and PS1 by activating PI3K/Akt pathway in AD.

MicroRNA-135b has a neuroprotective role via targeting of β-site APP-cleaving enzyme 1.

MicroRNAs (miRs) are a class of endogenous small non-coding RNAs that have been revealed to negatively mediate the expression of their target genes at the post-transcriptional level. Recently, particular miRs have demonstrated an involvement in the pathogenesis of Alzheimer's disease (AD). However, the specific role of miR-135b in AD has yet to be elucidated. The present study aimed to investigate the neuroprotective role of miR-135b, in addition to its underlying mechanism. Herein, reverse transcription-quantitative polymerase chain reaction was conducted to determine miR-135b expression levels in the peripheral blood samples of patients with AD and age-matched normal controls. The data of the present study revealed that the expression levels of miR-135b were significantly reduced in the peripheral blood of AD patients compared with normal controls (P<0.01). In vitro MTT analyses identified that the overexpression of miR-135b significantly enhanced the proliferation of hippocampal cells (P<0.01). Furthermore, in vivo analysis using a Y-maze test indicated that injection with miR-135b mimics into the third ventricle of anesthetized SAMP8 mice significantly enhanced their learning and memory capacities (P<0.01). Molecular mechanism investigations identified β-site APP-cleaving enzyme 1 (BACE1) as a direct target gene of miR-135b, and the latter was identified to negatively mediate the protein expression levels of BACE1 in hippocampal cells, in addition to hippocampal tissues, of SAMP8 mice. Based on the aforementioned findings, we propose that miR-135b has a neuroprotective role via direct targeting of BACE1 and, thus, may be used for the treatment of AD.

Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission.

Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer’s disease (AD)-associated amyloid βpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates.

BIN1 regulates BACE1 intracellular trafficking and amyloid-β production.

BIN1 is a genetic risk factor of late-onset Alzheimer disease (AD), which was identified in multiple genome-wide association studies. BIN1 is a member of the amphiphysin family of proteins, and contains N-terminal Bin-Amphiphysin-Rvs and C-terminal Src homology 3 domains. BIN1 is widely expressed in the mouse and human brains, and has been reported to function in the endocytosis and the endosomal sorting of membrane proteins. BACE1 is a type 1 transmembrane aspartyl protease expressed predominantly in neurons of the brain and responsible for the production of amyloid-β peptide (Aβ). Here we report that the depletion of BIN1 increases cellular BACE1 levels through impaired endosomal trafficking and reduces BACE1 lysosomal degradation, resulting in increased Aβ production. Our findings provide a mechanistic role of BIN1 in the pathogenesis of AD as a novel genetic regulator of BACE1 levels and Aβ production.

Gastrodin suppresses BACE1 expression under oxidative stress condition via inhibition of the PKR/eIF2α pathway in Alzheimer’s disease

The expression of β-site APP-cleaving enzyme 1 (BACE1) is increased in the brain of late-onset sporadic Alzheimer’s disease (AD) and oxidative stress may be the potential cause of this event. The phenolic glucoside gastrodin (Gas), a main component of a Chinese herbal medicine Gastrodia elata Blume, has been demonstrated to display antioxidant activity and suppresses BACE1 expression. However, the mechanisms by which Gas suppresses BACE1 expression are not clear. Morris water maze test was performed to assess the effect of Gas treatment on memory impairments in Tg2576 mice. The level of oxidative stress in the brain of Tg2576 mice was determined by measuring the superoxide dismutase (SOD) activity, catalase (CAT) activity, and the levels of malondialdehyde (MDA) and ROS. In vivo and in vitro, we detected the expression levels of BACE1, pPKRThr446, PKR, pPERKThr981, PERK, peIF2αSer51, and eIF2α using western blot analysis. We found that Gas improved learning and memory abilities of Tg2576 transgenic mice and attenuated intracellular oxidative stress in hippocampi of Tg2576 mice. We discovered that the expression levels of BACE1, activated PKR (pPKRThr446) and activated eIF2α (peIF2αSer51) were elevated in the brains of Tg2576 mice and hydrogen peroxide (H2O2)-stimulated SH-SY5Y cells. Moreover, peptide PKR inhibitor (PRI) and Gas down-regulated BACE1 expression in Tg2576 mice and H2O2-stimulated SH-SY5Y cells by inhibiting activation of PKR and eIF2α. Gas alleviates memory deficits in mice and suppresses BACE1 expression by inhibiting the protein kinase/Eukaryotic initiation factor-2α (PKR/eIF2α) pathway. The research suggested that Gas may develop as an drug candidate in neurodegenerative diseases.

Effects of hypoxia on the survival and Alzheimer's disease-related protein expression in HEK293 cells stably expression APP695 protein

To further study the regulation of hypoxia on Alzheimer's disease (AD) pathogenesis, we investigate the effect of hypoxia on the effect of cell survival and expression of related proteins in HEK293 cells stably expressing APP695 Swedish mutantK595N/M596L (HEK293-APP695 cells). HEK293-APP695 cells were cultured at hypoxia condition (0.3% O2). The survival rate of HEK293-APP695 cells was measured by CCK-8 assay. The protein expression levels of APP, APP-CTFs and BACE1 were detected by Western blot. The survival of HEK293-APP695 cells was obviously decreased after exposed to hypoxia. The expression of APP was reduced, and the expression of APP-CTFs was increased under hypoxia. Our data indicate that hypoxia accelerated cell death of HEK293-APP695 cells by increasing the cleavage of APP and production of β-secretase (β-site amyloid precursor protein cleavage enzyme1, BACE1).

Abstract 222: Hyperglycemia Reduces Levels of Soluble Amyloid Precursor Protein-α in Mouse Hippocampus and Cortex - Role of Cerebral Microvessels

In the present study, we tested hypothesis that hyperglycemia impairs the neuro-protective function of cerebral microvessels. Type 1 diabetes (T1D) was induced by treatment of mice with streptozotocin (blood glucose &gt;300 mg/dL for 2 months). Brain tissues and cerebral microvessels were isolated from diabetic and control mice. We observed significant decrease in the protein levels of soluble amyloid precursor protein-α (sAPPα, a product of α-cleavage of APP and a neurotrophic and neuroprotective molecule) in the total brain tissue of diabetic mice (n=5, P&lt;0.05). Further examination of brain regions revealed that the reduction of protein levels of sAPPα was only detected in hippocampus and cortex but not in cerebellum (n=6, P&lt;0.05). Our studies also revealed that there were no significant differences in the protein expression of APP and its non-amyloidogenic processing enzymes, α-secretases (a disintegrin and metalloprotease 10 [ADAM10], ADAM9, and ADAM17) in total brain tissue between T1D mice and control mice (n=5-6, P&gt;0.05). The protein level of β-processing enzyme BACE1 was also not changed in T1D mouse brain. In contrast, the protein levels of APP and ADAM10 were significantly decreased in cerebral microvasculature of T1D mice, as compared to control mice (n=9-10, P&lt;0.05). Immunofluorescent confocal microscopy also demonstrated that expressions of APP and ADAM10 in cerebral microvasculature (identified by co-staining with CD31) in hippocampus and cortex were reduced in T1D mice. Of note, cerebrovascular protein levels of ADAM9, ADAM17 and BACE1 were not affected by hyperglycemia (n=5-7, P&gt;0.05). These results suggest that hyperglycemia impairs APP expression and α-processing in brain vasculature, thereby leading to the reduction of brain content of sAPPα. The selective loss of neuro-protective molecule sAPPα in hippocampus and cortex may be an important mechanism contributing to the development of cognitive dysfunction in diabetes.

Long non-coding RNA BACE1-AS is a novel target for anisomycin-mediated suppression of ovarian cancer stem cell proliferation and invasion.

Human ovarian cancer stem cells (OCSCs) are one of the main factors affecting ovarian cancer cell metastasis, recurrence, prognosis and tolerance to chemotherapy drugs. However, the mechanisms of OCSC proliferation and invasion are not clear. Recent studies suggest that anisomycin can inhibit the proliferative and invasive ability of various tumor cells by increasing the production of the toxic amyloid β (Aβ1-42) peptides from the amyloid precursor protein (APP). We explored whether anisomycin could also suppress human OCSC proliferation and invasion. The CD44+/CD117+ OCSCs were enriched from human clinical ovarian tumor tissues. OCSCs treated with anisomycin showed reduced proliferation compared to controls. Moreover, anisomycin significantly suppressed the invasive capacity of OCSCs in vitro, as indicated by cell migration assays. The mRNA expression levels of long non-coding RNA (lncRNA) β-site APP cleaving enzyme 1 antisense strand (BACE1-AS) were significantly increased in anisomycin-treated OCSCs compared to controls. In addition, mRNA and protein levels of BACE1 and Aβ1-42 were increased in anisomycin-treated OCSCs compared to controls. We confirmed that anisomycin suppressed the growth of xenograft tumors formed by OCSCs in vivo. Finally, when expression of lncRNA BACE1-AS was silenced using siRNA, BACE1 expression was downregulated and the antiproliferative and anti-invasive effects of anisomycin were reduced. Overall, we identified lncRNA BACE1-AS as a novel target for anisomycin. Elevation of lncRNA BACE1-AS expression is a potential mechanism for suppressing human OCSC proliferation and invasion.

SEPT8 modulates β-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.

Dysfunction and loss of synapses are early pathogenic events in Alzheimer's disease. A central step in the generation of toxic amyloid-β (Aβ) peptides is the cleavage of amyloid precursor protein (APP) by β-site APP-cleaving enzyme (BACE1). Here, we have elucidated whether downregulation of septin (SEPT) protein family members, which are implicated in synaptic plasticity and vesicular trafficking, affects APP processing and Aβ generation. SEPT8 was found to reduce soluble APPβ and Aβ levels in neuronal cells through a post-translational mechanism leading to decreased levels of BACE1 protein. In the human temporal cortex, we identified alterations in the expression of specific SEPT8 transcript variants in a manner that correlated with Alzheimer's-disease-related neurofibrillary pathology. These changes were associated with altered β-secretase activity. We also discovered that the overexpression of a specific Alzheimer's-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aβ peptides in neuronal cells. These changes were related to an increased half-life of BACE1 and the localization of BACE1 in recycling endosomes. These data suggest that SEPT8 modulates β-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1.

Cholesterol Potentiates &amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt;-Amyloid Genesis in Cultured Human Umbilical Vein Endothelial Cells

Cerebral Amyloid Angiopathy (CAA) occurs commonly among the elderly and almost invariably in patients with Alzheimer’s Disease (AD). The β-amyloid peptides (Aβ) are produced via the amy-loidogenic processing of β-Amyloid Precursor Protein (APP) by β-secretase-1 (BACE1) and γ- secretase. Vascular endothelial cells are lately shown to possess the molecular machinery of Aβ production, which might participate in the development of CAA. Hypercholesterolemia is considered a risk factor for AD, whereas less is known if cholesterol may modulate endothelial Aβ production. In the present study we verified the amyloidogenic capability of Human Umbilical Vein Endothelial Cells (HUVECs) in vitro and explored the effect of cholesterol exposure on their amy-loidogenic potential. Cholesterol treatments at 12.5 and 25 mg/dL significantly elevated APP, BACE1 and APP β-CTF protein levels and β-site APP cleavage activity in cell lysates, and Aβ40 levels in culture medium. However, coincubation with cholesterol at 50 and 100 mg/dL attenuated the viability of the cultured cells and diminished their amyloidogenic capability. These findings suggest that high cholesterol exposure is stressful to vascular endothelial cells, and at a certain dosage range can promote an amyloidogenic response in these cells.

Deposition of BACE-1 Protein in the Brains of APP/PS1 Double Transgenic Mice.

The main causes of Alzheimer's disease remain elusive. Previous data have implicated the BACE-1 protein as a central player in the pathogenesis of Alzheimer's disease. However, many inhibitors of BACE-1 have failed during preclinical and clinical trials for AD treatment. Therefore, uncovering the exact role of BACE-1 in AD may have significant impact on the future development of therapeutic agents. Three- and six-month-old female APP/PS1 double transgenic mice were used to study abnormal accumulation of BACE-1 protein in brains of mice here. Immunofluorescence, immunohistochemistry, and western blot were performed to measure the distributing pattern and expression level of BACE-1. We found obvious BACE-1 protein accumulation in 3-month-old APP/PS1 mice, which had increased by the time of 6 months. Coimmunostaining results showed BACE-1 surrounded amyloid plaques in brain sections. The abnormal protein expression might not be attributable to the upregulation of BACE-1 protein, as no significant difference of protein expression was observed between wild-type and APP/PS1 mice. With antibodies against BACE-1 and CD31, we found a high immunoreactive density of BACE-1 protein on the outer layer of brain blood vessels. The aberrant distribution of BACE-1 in APP/PS1 mice suggests BACE-1 may be involved in the microvascular abnormality of AD.

Hydrogen Sulfide Selectively Inhibits γ-Secretase Activity and Decreases Mitochondrial Aβ Production in Neurons from APP/PS1 Transgenic Mice.

Hydrogen sulfide (H2S) is now considered to be a gasotransmitter and may be involved in the pathological process of Alzheimer's disease (AD). A majority of APP is associated with mitochondria and is a substrate for the mitochondrial γ-secretase. The mitochondria-associated APP metabolism where APP intracellular domains (AICD) and Aβ are generated locally and may contribute to mitochondrial dysfunction in AD. Here, we aimed to investigate the ability of H2S to mediate APP processing in mitochondria and assessed the possible mechanisms underlying H2S-mediated AD development. We treated neurons from APP/PS1 transgenic mice with a range of sodium hydrosulfide (NaHS) concentrations. NaHS attenuated APP processing and decreased Aβ production in mitochondria. Meanwhile, NaHS did not changed BACE-1 and ADAM10 (a disintegrin and metalloprotease 10) protein levels, but NaHS (30μM) significantly increased the levels of presenilin 1(PS1), PEN-2, and NCT, as well as improved the γ-secretase activity, while NaHS (50μM) exhibits the opposing effects. Furthermore, the intracellular ATP and the COX IV activity of APP/PS1 neurons were increased after 30μM NaHS treatment, while the ROS level was decreased and the MMP was stabilized. The effect of NaHS differs from DAPT (a non-selective γ-secretase inhibitor), and it selectively inhibited γ-secretase in vitro, without interacting with Notch and modulating its cleavage. The results indicated that NaHS decreases Aβ accumulation in mitochondria by selectively inhibiting γ-secretase. Thus, we provide a mechanistic view of NaHS is a potential anti-AD drug candidate and it may decrease Aβ deposition in mitochondria by selectively inhibiting γ-secretase activity and therefore protecting the mitochondrial function during AD conditions.

Bisecting GlcNAc modification stabilizes BACE1 protein under oxidative stress conditions.

β-Site amyloid precursor protein-cleaving enzyme-1 (BACE1) is a protease essential for amyloid-β (Aβ) production in Alzheimer's disease (AD). BACE1 protein is known to be up-regulated by oxidative stress-inducing stimuli but the mechanism for this up-regulation still needs to be clarified. We have recently found that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc) by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene) and that GnT-III deficiency reduces Aβ-plaque formation in the brain by accelerating lysosomal degradation of BACE1. Therefore, we hypothesized that bisecting GlcNAc would stabilize BACE1 protein on oxidative stress. In the present study, we first show that Aβ deposition in the mouse brain induces oxidative stress, together with an increase in levels of BACE1 and bisecting GlcNAc. Furthermore, prooxidant treatment induces expression of BACE1 protein in wild-type mouse embryonic fibroblasts (MEFs), whereas it reduces BACE1 protein in GnT-III (Mgat3) knock-out MEFs by accelerating lysosomal degradation of BACE1. We purified BACE1 from Neuro2A cells and performed LC/ESI/MS analysis for BACE1-derived glycopeptides and mapped bisecting GlcNAc-modified sites on BACE1. Point mutations at two N-glycosylation sites (Asn(153) and Asn(223)) abolish the bisecting GlcNAc modification on BACE1. These mutations almost cancelled the enhanced BACE1 degradation seen in Mgat3(-/-) MEFs, indicating that bisecting GlcNAc on BACE1 indeed regulates its degradation. Finally, we show that traumatic brain injury-induced BACE1 up-regulation is significantly suppressed in the Mgat3(-/-) brain. These results highlight the role of bisecting GlcNAc in oxidative stress-induced BACE1 expression and offer a novel glycan-targeted strategy for suppressing Aβ generation.

Tripchlorolide Attenuates β-amyloid Generation via Suppressing PPARγ-Regulated BACE1 Activity in N2a/APP695 Cells

Due to its apparent rate-limiting function, BACE1 (β-secretase) appears to be a prime target for prevention of amyloid-β (Aβ) generation in brains with Alzheimer's disease (AD). The activity of BACE1 is regulated by peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor binding site of the BACE1 promoter, indicating that PPARγ may be a potential target for AD treatment. Several studies have demonstrated that PPARγ activation is involved in the immunostimulation of amyloid-β precursor protein processing by nonsteroidal anti-inflammatory drugs (NSAIDs). The present study found that tripchlorolide (T4), with a similar chemical structure to that of NSAIDs, decreased the levels of Aβ secreted in N2a-APP695 cells. T4 treatment reduced the mRNA and protein levels of BACE1 and the protein level of sAPPβ, a cleaved N-terminal fragment of APP by BACE1. The treatment also translocated PPARγ from cytoplasm to nuclear. Intriguingly, T4, like pioglitazone (a PPARγ agonist), suppressed the BACE1 activity in N2a-APP695 cells, which was attenuated by GW9662 (a PPARγ antagonist). These results indicate that T4 may be a PPARγ agonist to enhance the binding of nuclear PPARγ to the BACE1 promoter, which may in turn inhibit the transcription and translation of BACE1, suppress the activity of BACE1, and ultimately attenuate the generation of Aβ. Due to its capability to alter Aβ generation and to protect central neural system against the neurotoxicity of Aβ, T4 may serve as a promising agent in modulating Aβ-related pathology in Alzheimer's disease.

Amyloid β-protein oligomers upregulate the β-secretase, BACE1, through a post-translational mechanism involving its altered subcellular distribution in neurons

Backgroundβ-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is a membrane-bound aspartyl protease that initiates amyloid β-protein (Aβ) generation. Aberrant elevation of BACE1 levels in brains of Alzheimer’s disease (AD) patients may involve Aβ. In the present study, we used a neuron culture model system to investigate the effects of Aβ on BACE1 expression as well as the underlying mechanisms.ResultsRat primary cortical neurons were treated with relatively low concentrations (2.5 μM) of Aβ42 oligomers (Aβ-O) or fibrils (Aβ-F) for 2–3 days. Aβ-O induced a significant increase in protein levels of BACE1, while Aβ-F only had a marginal effect. Levels of amyloid precursor protein (APP) and the major α-secretase, ADAM10, remained unaltered upon treatment with both types of Aβ. Aβ-O treatment resulted in activation of eIF2α and caspase 3 in a time-dependent manner, with no changes in the endoplasmic reticulum (ER) stress marker, GRP78, indicating that a typical ER stress response is not induced under our experimental conditions. Furthermore, Aβ-O did not affect BACE1 mRNA expression but augmented the levels of exogenous BACE1 expressed via recombinant adenoviruses, indicating regulation of BACE1 protein expression, not at the transcriptional or translational but the post-translational level. Immunocytochemical analysis revealed that Aβ-O causes a significant increase in BACE1 immunoreactivity in neurites (both axons and dendrites), but not soma of neurons; this change appears relevant to the mechanism of Aβ-O-induced BACE1 elevation, which may involve impairment of BACE1 trafficking and degradation. In contrast, Aβ-O had no effect on APP immunoreactivity.ConclusionOur results collectively suggest that Aβ oligomers induce BACE1 elevation via a post-translational mechanism involving its altered subcellular distribution in neurons, which possibly triggers a vicious cycle of Aβ generation, thus contributing to the pathogenetic mechanism of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0163-5) contains supplementary material, which is available to authorized users.

The Association of Amyloid-β Protein Precursor With α- and β-Secretases in Mouse Cerebral Cortex Synapses Is Altered in Early Alzheimer’s Disease

Amyloid-β peptides (Aβ), the proposed triggers of synaptic dysfunction in early Alzheimer's disease (AD), derive from the endoproteolytic cleavage of amyloid-β precursor protein (APP) by β-secretases (BACE1), whereas APP cleavage by α-secretases (ADAM10) abrogates Aβ formation. We now mapped the synaptic localization of APP, ADAM10, and BACE1 in the mouse cerebral cortex. All three proteins were present in cortical synapses and subsynaptic fractionation revealed that APP was located mainly in the pre-synaptic active zone (53%) and in the post-synaptic density (37%), whereas ADAM10 was enriched in the post-synaptic density (61%) and BACE1 was concentrated in extra-synaptic regions (72%). Immunocytochemistry analysis further showed that APP and BACE1 were co-localized in about 30% of both glutamatergic and GABAergic terminals, whereas few terminals were endowed with ADAM10. This distribution is modified in a mouse model of early AD based on Aβ1-42-intracerebroventricular injection, where the synaptic levels of APP and ADAM10 increased by 30%, whereas BACE1 levels were reduced. This suggests that, in early AD, there are compensatory mechanisms to avoid Aβ overload in cortical synapses favoring the non-amyloidogenic processing of APP.

Genetic Variation in δ-Opioid Receptor Associates with Increased β- and γ-Secretase Activity in the Late Stages of Alzheimer’s Disease

The agonist-induced activation of human δ-opioid receptor (δOR) has been shown to increase β- (BACE1) and γ-secretase activities leading to increased production of amyloid-β (Aβ) peptide. We have recently shown that phenylalanine to cysteine substitution at amino acid 27 in δOR (δOR-Phe27Cys) increases amyloid-β protein precursor processing through altered endocytic trafficking. Also, a genetic meta-analysis of the δOR-Phe27Cys variation (rs1042114) in two independent Alzheimer's disease (AD) patient cohorts indicated that the heterozygosity of δOR-Phe27Cys increases the risk of AD. Here, we investigated α-, β-, and γ-secretase activities in human brain with respect to δOR-Phe27Cys variation in the temporal cortex of 71 subjects with varying degree of AD-related neurofibrillary pathology (Braak stages I-VI). As a result, a significant increase in β- (p = 0.03) and γ- (p = 0.01), but not α-secretase, activities was observed in late stage AD samples (Braak stages V-VI), which were heterozygous for δOR-Phe27Cys as compared to the δOR-Phe27 and δOR-Cys27 homozygotes. The augmented β-secretase activity was not associated with increased mRNA expression or protein levels of BACE1 in the late stage AD patients, who were heterozygous for the δOR-Phe27Cys variation. These findings suggest that δOR-Phe27Cys variation modulates β- and γ-secretase activity in the late stages of AD likely via post-translational mechanisms other than alterations in the mRNA or protein levels of BACE1, or, in the expression of γ-secretase complex components.

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β-stimulated SK-N-SH cells.

Pomegranate fruit, Punica granatum L. (Punicaceae), and its constituents have been shown to inhibit inflammation. In this study, we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β-stimulated SK-N-SH cells. An enzyme immunoassay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β-stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB, and phospho-IKK proteins was evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. PWE (25-200μg/ml) dose dependently reduced COX-2-dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK was significantly (p<0.001) inhibited by PWE (50-200μg/ml). Our studies also show that PWE (50-200μg/ml) significantly (p<0.01) inhibited NF-κB transactivation in TNFα-stimulated HEK293 cells. Furthermore, PWE inhibited BACE-1 and Aβ expression in SK-N-SH cells treated with IL-1β. Taken together, our study demonstrates that pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders such as Alzheimer's disease.

Lamotrigine Reduces β-Site AβPP-Cleaving Enzyme 1 Protein Levels Through Induction of Autophagy.

Lamotrigine (LTG), a broad-spectrum anti-epileptic drug widely used in treatment for seizures, shows potential efficacy in Alzheimer's disease (AD) therapy. Chronic LTG treatment rescues the suppressed long-term potentiation, loss of spines and cognitive deficits in AβPP/PS1 mice, known to overexpress a chimeric mouse/human mutant amyloid-β protein precursor (AβPP) and a mutant human presenilin 1 (PS1). These changes are accompanied by reduction of amyloid-β (Aβ) plaques density and of levels of β-C-terminal fragment of AβPP (β-CTF), a fragment of AβPP cleaved by β-secretase. These results suggest LTG treatment reduces Aβ production, possibly through modulation of cleavage of AβPP by β-secretase. However, the underlying mechanisms still remain unclear. In this study, decreased protein levels, but not mRNA levels of β-site AβPP-cleaving enzyme 1 (BACE1), were observed in cultured HEK293 cells and the brains of AβPP/PS1 transgenic mice upon LTG treatment. Moreover, LTG treatment suppressed mammalian target of rapamycin (mTOR) signaling, while enhancing activation of cAMP response element binding protein (CREB), two signaling pathways essential for autophagy induction. LTG treatment increased the numbers of LC3-GFP+ puncta and LC3-II levels in HEK293 cells, indicating an induction of autophagy. The downregulation of BACE1 by LTG treatment was prevented by the autophagy inhibitor 3-Methyladenine. Therefore, this study shows that LTG treatment reduces the protein levels of BACE1 through activation of autophagy, possibly via inhibition of mTOR signaling and activation of CREB.

Disrupted-in-Schizophrenia-1 Attenuates Amyloid-β Generation and Cognitive Deficits in APP/PS1 Transgenic Mice by Reduction of β-Site APP-Cleaving Enzyme 1 Levels.

Disrupted-in-Schizophrenia-1 (DISC1) is a genetic risk factor for a wide range of major mental disorders, including schizophrenia, major depression, and bipolar disorders. Recent reports suggest a potential role of DISC1 in the pathogenesis of Alzheimer's disease (AD), by referring to an interaction between DISC1 and amyloid precursor protein (APP), and to an association of a single-nucleotide polymorphism in a DISC1 intron and late onset of AD. However, the function of DISC1 in AD remains unknown. In this study, decreased levels of DISC1 were observed in the cortex and hippocampus of 8-month-old APP/PS1 transgenic mice, an animal model of AD. Overexpression of DISC1 reduced, whereas knockdown of DISC1 increased protein levels, but not mRNA levels of β-site APP-Cleaving Enzyme 1 (BACE1), a key enzyme in amyloid-β (Aβ) generation. Reduction of BACE1 protein levels by overexpression of DISC1 was accompanied by an accelerating decline rate of BACE1, and was blocked by the lysosomal inhibitor chloroquine, rather than proteasome inhibitor MG-132. Moreover, overexpression of DISC1 in the hippocampus with an adeno-associated virus reduced the levels of BACE1, soluble Aβ40/42, amyloid plaque density, and rescued cognitive deficits of APP/PS1 transgenic mice. These results indicate that DISC1 attenuates Aβ generation and cognitive deficits of APP/PS1 transgenic mice through promoting lysosomal degradation of BACE1. Our findings provide new insights into the role of DISC1 in AD pathogenesis and link a potential function of DISC1 to the psychiatric symptoms of AD.