BACE1 Protein Research Articles

Abstract TP486: Role of Beta-Site Amyloid Precursor Protein-Cleaving Enzyme 2 in the Metabolism of Amyloid Precursor Protein in Human Brain Microvascular Endothelial Cells

Under physiological conditions, β-site amyloid precursor protein (APP)-cleaving enzyme 2 (BACE2) cleaves APP within Aβ sequence thereby functioning like a α-secretase. However, BACE2 could also function as a conditional β-secretase during aging, contributing to Alzheimer’s disease pathogenesis. To date the physiological functions of BACE2 in endothelium are largely unknown. The present study is therefore designed to investigate the role of BACE2 in APP metabolism in human BMECs. Cultured human BMECs (passage 5-6 or passage 22) were treated with BACE2siRNA (30 nM, for 3 days), levels of soluble APPα (sAPPα, a neurotrophic product of non-amyloidogenic processing of APP) and Aβ40 in the supernatant were measured. In human BMECs (passage 5-6), genetic inactivation of BACE2 significantly decreased production of sAPPα (n=12, P<0.05), but had no effect on production of Aβ40 (n=9, P>0.05). BACE2siRNA treatment significantly suppressed APP protein expression (n=7, P<0.05), but augmented protein levels of BACE1 (n=7, P<0.05). Genetic inactivation of BACE2 did not change protein levels of mature ADAM10 (n=7, P>0.05). Thus, reduced sAPPα secretion by BACE2siRNA treatment is likely caused not only by decreased α-secretase-like function of BACE2, but also by reduced APP expression. We further examined the effects of BACE2siRNA in senescent human BMECs. In cultured human BMECs (passage 22), protein expressions of senescent markers (p 21Cip1 and p 16INK4a ) were significantly increased (n=4, P<0.05). Genetic inactivation of BACE2 in senescent human BMECs also significantly suppressed secretion of sAPPα (n=8, P<0.05), but did not affect Aβ40 production (n=8, P>0.05). BACE2-siRNA treatment significantly inhibited protein expressions of APP and mature ADAM10 (n=7, P<0.05), but did not change BACE1 protein expression (n=7, P>0.05). Thus in senescent human BMECs, reduced APP expression and impaired α-processing may play important roles in the decreased sAPPα production. Since our previous studies have demonstrated that endothelial production of sAPPα significantly contributes to the sAPPα content in the hippocampus, our current findings suggests that inhibition of BACE2 could impair protective function of sAPPα in the hippocampus.

BACE1 partial deletion induces synaptic plasticity deficit in adult mice

BACE1 is the first enzyme involved in APP processing, thus it is a strong therapeutic target candidate for Alzheimer’s disease. The observation of deleterious phenotypes in BACE1 Knock-out (KO) mouse models (germline and conditional) raised some concerns on the safety and tolerability of BACE1 inhibition. Here, we have employed a tamoxifen inducible BACE1 conditional Knock-out (cKO) mouse model to achieve a controlled partial depletion of BACE1 in adult mice. Biochemical and behavioural characterization was performed at two time points: 4–5 months (young mice) and 12–13 months (aged mice). A ~50% to ~70% BACE1 protein reduction in hippocampus and cortex, respectively, induced a significant reduction of BACE1 substrates processing and decrease of Aβx-40 levels at both ages. Hippocampal axonal guidance and peripheral nerve myelination were not affected. Aged mice displayed a CA1 long-term potentiation (LTP) deficit that was not associated with memory impairment. Our findings indicate that numerous phenotypes observed in germline BACE1 KO reflect a fundamental role of BACE1 during development while other phenotypes, observed in adult cKO, may be absent when partially rather than completely deleting BACE1. However, we demonstrated that partial depletion of BACE1 still induces CA1 LTP impairment, supporting a role of BACE1 in synaptic plasticity in adulthood.

Silencing of long noncoding RNA XIST attenuated Alzheimer's disease-related BACE1 alteration through miR-124.

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. However, its pathogeneticmechanism is still poorly understood. An increasing number of studies have evidenced the important role of long noncoding RNAs (lncRNAs) in AD. The aim of the current study was to investigate the effect and molecular mechanism of the lncRNA X-inactive specific transcript (XIST) in AD. Bilateral common carotid arteryocclusion (2VO) was used to induce an AD model in mice. Hydrogen peroxide (H2 O2 ) was used to induce an AD model in N2a cells. The lncRNA XIST, miR-124, and BACE1 messenger RNA expression levels were detected by a real-time polymerase chain reaction. The BACE1 protein expression level was detected by western blot and immunofluorescence assay. The Aβ1-42 expression level was detected using an enzyme-linked immunosorbent assay kit. The expression level of lncRNA XIST was significantly upregulated in AD models, both in vivo and in vitro. Silencing of lncRNA XIST negatively regulated miR-124 and positively regulated BACE1 expression in N2a cells, which is attenuated by cotransfection of anti-miR-124 oligodeoxyribonucleotide (AMO-124). Silencing of lncRNA XIST reversed the effect of H2 O2 on miR-124, BACE1, and Aβ1-42 expression in N2a cells, which was reversed by cotransfection of AMO-124. Silencing of lncRNA XIST attenuated AD-related BACE1 alteration through miR-124. LncRNA XIST may be a new potential target for the treatment of AD.

A Curcumin Analog Reduces Levels of the Alzheimer's Disease-Associated Amyloid-β Protein by Modulating AβPP Processing and Autophagy.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with no cure currently available. A pathological hallmark of AD is accumulation and deposition of amyloid-β protein (Aβ), a ∼4 kDa peptide generated through serial cleavage of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. Curcumin is a natural compound primarily found in the widely used culinary spice, turmeric, which displays therapeutic potential for AD. Recently, we reported the development of curcumin analogs and identified a lead compound, curcumin-like compound-R17 (CLC-R17), that significantly attenuates Aβ deposition in an AD transgenic mouse model. Here, we elucidated the mechanisms of this analog on Aβ levels and AβPP processing using cell models of AD. Using biochemical methods and our recently developed nanoplasmonic fiber tip probe technology, we showed that the lead compound potently lowers Aβ levels in conditioned media and reduces oligomeric amyloid levels in the cells. Furthermore, like curcumin, the lead compound attenuates the maturation of AβPP in the secretory pathway. Interestingly, it upregulated α-secretase processing of AβPP and inhibited β-secretase processing of AβPP by decreasing BACE1 protein levels. Collectively, our data reveal mechanisms of a promising curcumin analog in reducing Aβ levels, which strongly support its development as a potential therapeutic for AD.

Distinct anterograde trafficking pathways of BACE1 and amyloid precursor protein from the TGN and the regulation of amyloid-β production.

Processing of amyloid precursor protein (APP) by the β-secretase BACE1 is the initial step of the amyloidogenic pathway to generate amyloid-β (Aβ). Although newly synthesized BACE1 and APP are transported along the secretory pathway, it is not known whether BACE1 and APP share the same post-Golgi trafficking pathways or are partitioned into different transport routes. Here we demonstrate that BACE1 exits the Golgi in HeLa cells and primary neurons by a pathway distinct from the trafficking pathway for APP. By using the Retention Using Selective Hooks system, we show that BACE1 is transported from the trans-Golgi network to the plasma membrane in an AP-1- and Arf1/4-dependent manner. Subsequently, BACE1 is endocytosed to early and recycling endosomes. Perturbation of BACE1 post-Golgi trafficking results in an increase in BACE1 cleavage of APP and increased production of both Aβ40 and Aβ42. These findings reveal that Golgi exit of BACE1 and APP in primary neurons is tightly regulated, resulting in their segregation along different transport routes, which limits APP processing.

The effect of BACE1-AS on \u03b2-amyloid generation by regulating BACE1 mRNA expression

BackgroundThe BACE1 antisense transcript (BACE1-AS) is a conserved long noncoding RNA (lncRNA). The level of BACE1-AS is significantly increased and the level of the BACE1 mRNA is slightly increased in subjects with AD. BACE1-AS exerts a significant moderating effect on the expression of the BACE1 mRNA and promotes the formation of Aβ. After the administration of Aβ1-42 to SH-SY5Y cells and C57/BL6J mice, we detected the expression of BACE1-AS, BACE1 mRNA, and BACE1 protein, as well as the concentration of Aβ1-40. Then, we silenced the expression of BACE1-AS in SH-SY5Y and 20E2 cells using siRNAs targeting BACE1-AS and detected its effects on the levels of the BACE1 mRNA and BACE1 protein and Aβ1-40 generation.ResultsThe administration of Aβ1-42 increased the expression of BACE1-AS, BACE1 mRNA and protein, as well as the concentration of Aβ1-40 in SH-SY5Y cells and the brains of C57BL/6J mice. Pretreatment with the BACE1-AS siRNA inhibited the effect of Aβ1-42 on increasing the expression of BACE1-AS and BACE1, as well as the generation of Aβ.ConclusionsThe mechanism by which exogenous Aβ1-42 induces BACE1 expression and Aβ generation is mediated by BACE1-AS. BACE1-AS is involved in the mechanism regulating BACE1 expression and Aβ generation in APPsw transgenic cells.

The BACE1-Specific DNA Aptamer A1 Rescues Amyloid-β Pathology and Behavioral Deficits in a Mouse Model of Alzheimer's Disease.

Amyloid-β (Aβ) plaque deposits in the brain are considered to be one of the main pathological markers of Alzheimer's disease (AD). The sequential proteolytic cleavage of amyloid precursor protein (APP) by the aspartyl proteases β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase produces Aβ. Therefore, BACE1 inhibition is a very attractive target for the treatment of AD. Our previous work identified a DNA aptamer named A1 that can bind to BACE1 with high affinity and specificity and exhibits a distinct inhibitory effect on BACE1 activity in an AD cell model. The purpose of this research was to test the effect of aptamer A1 in Tg6799 mice. Four-month-old Tg6799 mice were randomly divided into two groups and treated with aptamer A1 and ineffective aptamer A1scr, respectively, by intracerebroventricular injection. Subsequent behavioral experiments showed that treatment with the aptamer A1 improved the cognitive abilities of the AD mice. Western blot indicated that BACE1 and soluble amyloid precursor protein β (sAPPβ) expression significantly decreased in the A1-treated mice. Moreover, aptamer A1 reduced the content of Aβ42 and the number and density of senile plaques in AD mice. Therefore, our results indicate that aptamer A1 is a novel specific and potent BACE1 inhibitor and is a promising potential target for the treatment of AD.

Multiple BACE1 inhibitors abnormally increase the BACE1 protein level in neurons by prolonging its half-life

IntroductionThere is keen interest in elucidating the biological mechanisms underlying recent failures of β-site amyloid precursor protein–cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. MethodsWe developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. ResultsSeven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid β (Aβ). DiscussionElevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such β-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aβ by dissociating β/γ secretase complexes are better candidates because they neither inhibit β- and γ-secretase nor increase BACE1 levels.

11β-HSD1 Inhibition Rescues SAMP8 Cognitive Impairment Induced by Metabolic Stress.

Ageing and obesity have been shown to increase the risk of cognitive decline and Alzheimer's disease (AD). Besides, elevated glucocorticoid (GCs) levels cause metabolic stress and have been associated with the neurodegenerative process. Direct pieces of evidence link the reduction of GCs caused by the inhibition of 11β-HSD type 1 (11β-HSD1) with cognitive improvement. In the present study, we investigated the beneficial effects of 11β-HSD1 inhibitor (i) RL-118 after high-fat diet (HFD) treatment in the senescence-accelerated mouse prone 8 (SAMP8). We found an improvement in glucose intolerance induced by HFD in mice treated with RL-118, a significant reduction in 11β-HSD1 and glucocorticoid receptor (GR) protein levels. Furthermore, specific modifications in the FGF21 activation after treatment with 11β-HSD1i, RL-118, which induced changes in SIRT1/PGC1α/AMPKα pathway, were found. Oxidative stress (OS) and reactive oxygen species (ROS), as well as inflammatory markers and microglial activation, were significantly diminished in HFD mice treated with 11β-HSD1i. Remarkably, treatment with 11β-HSD1i altered PERK pathway in both diet groups, increasing autophagy only in HFD mice group. After RL-118 treatment, a decrease in glycogen synthase kinase 3 (GSK3β) activation, Tau hyperphosphorylation, BACE1 protein levels and the product β-CTF were found. Increases in the non-amyloidogenic secretase ADAM10 protein levels and the product sAPPα were found in both treated mice, regardless of the diet. Consequently, beneficial effects on social behaviour and cognitive performance were found in treated mice. Thus, our results support the therapeutic strategy of selective 11β-HSD1i for the treatment of age-related cognitive decline and AD.

Pioglitazone Reduces β Amyloid Levels via Inhibition of PPARγ Phosphorylation in a Neuronal Model of Alzheimer's Disease.

It has been demonstrated that peroxisome proliferator-activated receptor γ (PPARγ) can regulate the transcription of its target gene, insulin-degrading enzyme (IDE), and thus enhance the expression of the IDE protein. The protein can degrade β amyloid (Aβ), a core pathological product of Alzheimer’s disease (AD). PPARγ can also regulate the transcription of other target gene, β-amyloid cleavage enzyme 1 (BACE1), and thus inhibit the expression of the BACE1 protein. BACE1 can hydrolyze amyloid precursor protein (APP), the precursor of Aβ. In adipose tissue, PPARγ agonists can inhibit the phosphorylation of PPARγ by inhibiting cyclin-dependent kinase 5 (CDK5), which in turn affects the expression of target genes regulated by PPARγ. PPARγ agonists may also exert inhibitory effects on the phosphorylation of PPARγ in the brain, thereby affecting the expression of the aforementioned PPARγ target genes and reducing Aβ levels. The present study confirmed this hypothesis by showing that PPARγ agonist pioglitazone attenuated the neuronal apoptosis of primary rat hippocampal neurons induced by Aβ1–42, downregulated CDK5 expression, weakened the binding of CDK5 to PPARγ, reduced PPARγ phosphorylation, increased the expression of PPARγ and IDE, decreased the expression of BACE1, reduced APP production, and downregulated intraneuronal Aβ1–42 levels. These effects were inhibited by the PPARγ antagonist GW9662. After CDK5 silencing with CDK5 shRNA, the above effect of pioglitazone was not observed, except when upregulating the expression of PPARγ in Aβ1–42 treated neurons. In conclusion, this study demonstrated that pioglitazone could inhibit the phosphorylation of PPARγ in vitro by inhibiting CDK5 expression, which in turn affected the expression of PPARγ target genes Ide and Bace1, thereby promoting Aβ degradation and reducing Aβ production. This reduced Aβ levels in the brain, thereby exerting neuroprotective effects in an AD model.

Characterization of Cerebrospinal Fluid BACE1 Species.

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the main brain β-secretase responsible for the amyloidogenic processing of the amyloid precursor protein (APP). Previous studies have suggested that cerebrospinal fluid (CSF) β-secretase activity may be a candidate diagnostic biomarker for Alzheimer's disease (AD), but biochemical characterization of BACE1 protein in CSF is needed. CSF samples from 19 AD patients and 19 age-matched non-AD controls (n = 19) were classified according to their Aβ42, total tau, and P-tau CSF biomarker levels. We found that β-secretase activity was higher in the CSF of AD subjects than in that of the controls. We found that the majority of the β-secretase activity in the CSF, measured using a peptide substrate homologous to the BACE1 cleavage site, was not inhibited by specific BACE1 inhibitors. We defined enzymatic activity attributable specifically to BACE1 as the activity that was blocked by the specific inhibitors, which is still higher in AD subjects. BACE1 protein levels were characterized by lectin binding, immunoprecipitation, blue native-PAGE, and western blotting using antibodies against specific protein domains. BACE1 was found to be present in human CSF as a mature form of ~ 70kDa that probably comprised truncated and full-length species, and also as an immature form of ~ 50kDa that retains the prodomain. CSF-BACE1 was found to assemble into hetero-complexes containing distinct species. Immunoblotting with an antibody against the C-terminus of BACE1 revealed significantly higher levels of the 70-kDa full-length BACE1, while the 50kDa immature form remained unaltered. When the 70-kDa species was probed with an antibody against the N-terminus of BACE1 (which does not discriminate between truncated and full-length forms), no increase in immunoreactivity was observed, suggesting that truncated forms of BACE1 do not increase in AD. In conclusion, the complexity of BACE1 species in CSF has to be taken into consideration when determining BACE1 activity and protein levels in CSF as biomarkers of AD.

QSAR Classification Models for Predicting the Activity of Inhibitors of Beta-Secretase (BACE1) Associated with Alzheimer\u2019s Disease

Alzheimer’s disease is one of the most common neurodegenerative disorders in elder population. The β-site amyloid cleavage enzyme 1 (BACE1) is the major constituent of amyloid plaques and plays a central role in this brain pathogenesis, thus it constitutes an auspicious pharmacological target for its treatment. In this paper, a QSAR model for identification of potential inhibitors of BACE1 protein is designed by using classification methods. For building this model, a database with 215 molecules collected from different sources has been assembled. This dataset contains diverse compounds with different scaffolds and physical-chemical properties, covering a wide chemical space in the drug-like range. The most distinctive aspect of the applied QSAR strategy is the combination of hybridization with backward elimination of models, which contributes to improve the quality of the final QSAR model. Another relevant step is the visual analysis of the molecular descriptors that allows guaranteeing the absence of information redundancy in the model. The QSAR model performances have been assessed by traditional metrics, and the final proposed model has low cardinality, and reaches a high percentage of chemical compounds correctly classified.

Electroacupuncture Mitigates Hippocampal Cognitive Impairments by Reducing BACE1 Deposition and Activating PKA in APP/PS1 Double Transgenic Mice.

Increased amyloid-β (Aβ) plaque deposition is thought to be the main cause of Alzheimer's disease (AD). β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is the key protein involved in Aβ peptide generation. Excessive expression of BACE1 might cause overproduction of neurotoxins in the central nervous system. Previous studies indicated that BACE1 initially cleaves the amyloid precursor protein (APP) and may subsequently interfere with physiological functions of proteins such as PKA, which is recognized to be closely associated with long-term potentiation (LTP) level and can effectively ameliorate cognitive impairments. Therefore, revealing the underlying mechanism of BACE1 in the pathogenesis of AD might have a significant impact on the future development of therapeutic agents targeting dementia. This study examined the effects of electroacupuncture (EA) stimulation on BACE1, APP, and p-PKA protein levels in hippocampal tissue samples. Memory and learning abilities were assessed using the Morris water maze test after EA intervention. Immunofluorescence, immunohistochemistry, and western blot were employed to assess the distribution patterns and expression levels of BACE1, APP, and p-PKA, respectively. The results showed the downregulation of BACE1 and APP and the activation of PKA by EA. In summary, EA treatment might reduce BACE1 deposition in APP/PS1 transgenic mice and regulate PKA and its associated substrates, such as LTP to change memory and learning abilities.

Palmitic Acid-Induced NAD+ Depletion is Associated with the Reduced Function of SIRT1 and Increased Expression of BACE1 in Hippocampal Neurons.

Increased levels of circulating fatty acids, such as palmitic acid (PA), are associated with the development of obesity, insulin resistance, type-2 diabetes and metabolic syndrome. Furthermore, these diseases are linked to an increased risk of cancer, cardiovascular diseases, mild cognitive impairment and even Alzheimer's disease (AD). However, the precise actions of elevated PA levels on neurons and their association with neuronal metabolic disruption that leads to the expression of pathological markers of AD, such as the overproduction and accumulation of the amyloid-β peptide, represent an area of intense investigation. A possible molecular mechanism involved in the effects of PA may be through dysfunction of the NAD+ sensor enzyme, SIRT1. Therefore, the aim of the present study was to analyze the relationship between the effects of PA metabolism on the function of SIRT1 and the upregulation of BACE1 in cultured hippocampal neurons. PA reduced the total amount of NAD+ in neurons that caused an increase in p65 K310 acetylation due to inhibition of SIRT1 activity and low protein content. Furthermore, BACE1 protein and its activity were increased, and BACE1 was relocated in neurites after PA exposure.

MicroRNA‐195 Alleviates Cognitive Impairment in Rat following Chronic Brain Hypoperfusion

ObjectiveTo observe whether chronic brain hypoperfusion (CBH) induced cognitive decline is associated with amyloid‐β (Aβ)‐cascade response process, dendritic degeneration and neuron death in the hippocampus as well as septal‐hippocampal cholinergic circuit disorder.Methods and resultsWe found that bilateral common carotid artery occlusion (2VO) generated CBH could induce significant cognitive decline and decreased fEPSP of septal‐hippocampal cholinergic circuit in rats, assessed by Morris water maze and in vivo electrophysiological recording. Subsequently, we found the overexpressed protein levels of amyloid precursor protein (APP) and β‐site APP cleaving enzyme 1 (BACE1), death receptor 6 (DR6), together with an activated cdk5/p25, glycogen synthase kinase 3β (GSK3β), inactivated protein phosphatase 2A, degenerated dendrites and neuron death in the hippocampus, as evaluated by western blotting, Golgi, TUNEL and immunofluorescence staining. Interestingly, microRNA‐195 (miR‐195) was found decreased in both hippocampus and basal forebrain. By bioinformatic prediction, we found that miR‐195 could target on 3’‐untranslated regions of Aβ cascade response related genes, including APP, BACE1, Cdk5r1, PME‐1 and Death receptor 6. Further study revealed that miR‐ 195 regulated the expression of APP, BACE1, p35, methylesterase (PME‐1) and DR6 protein at the post‐transcriptional level, as assessed by Dual luciferase reporter assay, siRNA and miR‐masking techniques. In vivo, knockdown of endogenous miR‐195 by lentiviral vector‐mediated over‐expression of its antisense molecule (lenti‐pre‐AMO‐195) elicited the phenotype of dementia and upregulated protein levels of APP, BACE1, p35, PME‐1 and DR6 in rats, whereas over‐expression of miR‐195 using lenti‐pre‐miR‐195 reduced dementia‐vulnerability and those protein levels triggered by CBH. Additionally, chromatin immunoprecipitation analysis showed that NFκB was bound to the promoter region of miR‐195 and inhibited its expression.ConclusionCBH could induce cognitive impairment involving Aβ aggregation, hyperphosphorylation of Tau, GSK3β activation, protein phosphatase‐2A inactivation, degeneration of dendrites and neuron death, which is associated with chronic brain hypoxia‐activated NFκB that inhibited endogenous biogenesis of miR‐195. Down‐regulation of miR‐195 up‐regulated the expression of a battery of Aβ cascade response related proteins at the post‐transcriptional level. MiR‐195 may play a key role in determining dementia susceptibility in CBH rats through a multi‐target way at the post‐transcriptional level, and exogenous complement of miR‐195 may be a potentially valuable anti‐dementia approach.Support or Funding InformationFoundation: This work was supported by Natural Science Foundation of China (81271207, 81070882, 81000499, 81471115, 81671052 and 81371211).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute exercise and brain BACE1 protein content: a time course study.

Obesity and insulin resistance are risk factors in the development of neurodegenerative disorders. Previous work suggests that one acute bout of exercise may have beneficial neuro‐protective effects in obese mice. The rate limiting enzyme in the production of amyloid‐beta peptides, BACE1, was reduced in the prefrontal cortex 2 h post‐exercise, however if these effects remain over time is unknown. We aimed to determine how long exercise–induced alterations persist in the prefrontal cortex and hippocampus following a single exercise bout. Male C57BL/6J mice were fed either a low (LFD, 10% kcals from lard) or a high fat diet (HFD, 60% kcals from lard) for 7 weeks. HFD mice then underwent an acute bout of treadmill running (15 m/min, 5% incline, 120 min) followed by 2‐, 8‐, or 24‐h of recovery. The HFD increased body mass (LFD 27.8 ± 1.05 vs. HFD 41.7 ± 0.60 g; P < 0.05) and glucose intolerance (AUC LFD 63.27 ± 4.5 vs. HFD 128.9 ± 4.6; P < 0.05). Prefrontal cortex BACE1 content was reduced 2‐ and 8‐h post‐exercise compared to sedentary HFD mice, however BACE1 protein content at 24 h was not different. Hippocampal BACE1 content was reduced 8‐ and 24‐h post‐exercise. Compared to the LFD, the HFD had higher prefrontal cortex phosphorylation of p38, JNK, and AMPK, indicative of increased neuronal stress. Post–exercise prefrontal cortex p38 and JNK phosphorylation were no different between the HFD or LFD groups, while ERK phosphorylation was significantly reduced by 24 h. The HFD increased JNK phosphorylation in the hippocampus. These results demonstrate the direct and potent effects of exercise on reducing BACE1 prefrontal cortex and hippocampal content. However the reduction in prefrontal cortex BACE1 content is short lived.

Astrocytes infected with Chlamydia pneumoniae demonstrate altered expression and activity of secretases involved in the generation of \u03b2-amyloid found in Alzheimer disease

BackgroundEpidemiologic studies strongly suggest that the pathophysiology of late-onset Alzheimer disease (AD) versus early-onset AD has environmental rather than genetic causes, thus revealing potentially novel therapeutic targets to limit disease progression. Several studies supporting the “pathogen hypothesis” of AD demonstrate a strong association between pathogens and the production of β-amyloid, the pathologic hallmark of AD. Although the mechanism of pathogen-induced neurodegeneration of AD remains unclear, astrocytes, a key player of the CNS innate immune response and producer/metabolizer of β-amyloid, have been implicated. We hypothesized that Chlamydia pneumoniae infection of human astrocytes alters the expression of the amyloid precursor protein (APP)-processing secretases, ADAM10, BACE1, and PSEN1, to promote β-amyloid formation. Utilizing immunofluorescent microscopy, molecular, and biochemical approaches, these studies explore the role of an intracellular respiratory pathogen, Chlamydia pneumoniae, as an environmental trigger for AD pathology. Human astrocytoma cells in vitro were infected with Chlamydia pneumoniae over the course of 6–72 h. The gene and protein expression, as well as the enzymatic activity of non-amyloidogenic (ADAM10), and pro-amyloidogenic (BACE1 and PSEN1) secretases were qualitatively and quantitatively assessed. In addition, the formation of toxic amyloid products as an outcome of pro-amyloidogenic APP processing was evaluated through various modalities.ResultsChlamydia pneumoniae infection of human astrocytoma cells promoted the transcriptional upregulation of numerous genes implicated in host neuroinflammation, lipid homeostasis, microtubule function, and APP processing. Relative to that of uninfected astrocytes, BACE1 and PSEN1 protein levels were enhanced by nearly twofold at 48–72 h post-Chlamydia pneumoniae infection. The processing of APP in Chlamydia pneumoniae-infected astrocytes favors the pro-amyloidogenic pathway, as demonstrated by an increase in enzymatic activity of BACE1, while that of ADAM10 was decreased. Fluorescence intensity of β-amyloid and ELISA-quantified levels of soluble-APP by products revealed temporally similar increases, confirming a BACE1/PSEN1-mediated processing of APP.ConclusionsOur findings suggest that Chlamydia pneumoniae infection of human astrocytes promotes the pro-amyloidogenic pathway of APP processing through the upregulation of expression and activity of β-secretase, upregulated expression of γ-secretase, and decreased activity of α-secretase. These effects of astrocyte infection provide evidence for a direct link between Chlamydia pneumoniae and AD pathology.

MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation.

MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (β-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-β precursor protein processing, amyloid-β load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.

French maritime pine bark treatment decelerates plaque development and improves spatial memory in Alzheimer's disease mice

BackgroundPlant extracts are increasingly investigated as potential drugs against Alzheimer's disease (AD) and dementia in general. Pycnogenol is an extract from the bark of the French maritime pine (Pinus pinaster Aiton subsp. atlantica) with known anti-oxidative and neuroprotective effects. Hypothesis/PurposePycnogenol is thought to improve cognitive functions in elderly. We wanted to investigate and quantify these effects in a model system of cerebral ß-amyloidosis/AD. Study design/methodsThis study experimentally assessed the effects of Pycnogenol on AD-related pathology in a ß-amyloidosis mouse model. APP-transgenic mice and controls were treated orally in a pre-onset and post-onset treatment paradigm. The effects of Pycnogenol were characterized by analysing ß-amyloid (Aß) plaques, number of neurons, glia coverage, myelination pattern, and cortical coverage with axons using immunohistochemistry. Aß levels were quantified using ELISA and gene expression levels of APP-processing enzymes ADAM10, BACE1 and IDE protein levels were determined by Western blot. Behavioural changes in circadian rhythm were monitored and spatial memory / cognition was assessed using a water maze test. ResultsPycnogenol significantly decreased the number of plaques in both treatment paradigms but did not alter levels of soluble Aß or the gene expression of APP-processing enzymes. The morphological analyses revealed no changes in the number of neurons, astrocytes, microglia, the myelination pattern, or the morphology of axons. Behavioural testing revealed an improvement of the spatial memory in the pre-onset treatment paradigm only. ConclusionOur results suggest to evaluate clinically a potential use of Pycnogenol in the prevention or in early stages of mild cognitive impairment (MCI) and AD.

PO-123 Voluntary wheel training attenuate brain aging in SAMP8 mice

Objective Alzheimer's disease (AD), is a progressive neurodegenerative disease. The pathological hallmarks including the deposition of senile plaque and neurofibrillary tangles (NFTs) in AD. The underlying molecular mechanisms for AD are not fully understood. Studies have shown that exercise as an economical and popular fitness method, long-term exercise can reduce the risk of AD, delay the occurrence of AD or delay the progression of AD. This study was to investigate the protective effect of the Voluntary wheel on cerebral cortex senescence in SAMP8 mice.&#x0D; Methods Fifteen SPF-class 7-month-old SAMP8 mice were randomly divided into control group (CON), voluntary wheel group (V) and voluntary wheel with chloroquine group (VQ), with 5 mice in each group. Each group was under adaptive feeding for one week. After 3 days of adaptive voluntary wheel training for V and VQ groups, the experiment officially started. The V and VQ groups performed an 8-week voluntary wheel training and the VQ group received a daily intraperitoneal injection of chloroquine (40 mg/kg). After 8 weeks intervention, the Morris water maze test was used to determine the changes in spatial learning and memory ability of each group. After 24 hours of the end of this experiment, the mice were killed by breaking neck. The degree of hippocampal cell injury was detected by Nissl staining; the expression of apoptosis and senescence-associated protein in the cerebral cortex was detected by Western blot.&#x0D; Results Compared with the CON group, the escape latency of the V group mice in the Morris water maze test was significantly shorter (P&lt;0.01), the number of crossing target quadrants was significantly increased (P&lt;0.05), however, the VQ group exacerbate above response compared with CON group. Nissl staining showed that the cells of cerebral cortex in the CON group had obvious damage, while the V group showed significant improvement. The VQ group had a significant damage compared with the V group; Western blot results showed that the expression of AD -like pathological changes such as BACE1 protein in the cortex of group V was significantly decreased, and the expression of P-GSK-3β protein was increased (P&lt;0.01) compared with CON group. On the contrary, compared with V group, VQ alleviated the improvement effect of voluntary running. (P&lt;0.05); the expression of apoptosis-related protein increased, and thedecreased in CON group , while voluntary running intervention down-regulated BAX, Cleaved-caspase3 , increased the Bcl-2 protein level compared with the CON group (P&lt;0.05). Furthermore, compared with the CON group, the expression level of aging-related protein AC-P53 was decreased in the V group, and the expression level of Sirt1 was increased. &#x0D; Conclusions The 8-week voluntary wheel training can significantly improve the learning and memory ability of SAMP8. Mechanismly, voluntary wheel training can improve the senescence of cerebral cortex by inhibiting cell apoptosis, the expression of AD –like pathological protein and increasing Sirt1 protein level.