BACE1 Protein Levels Research Articles

Mitigating Effect of Korea Red Ginseng Against Multi‐Tissue Iipotoxicity Caused by High‐Fat Diet Feeding

BackgroundsExcessive consumption of fat and its aberrant deposit in the body has been linked to the low‐grade inflammation in various susceptible organs and eventually causes tissue toxicity. This study examines the multifaceted suppressive effects of Korean Red ginseng extract (KRG) against high‐fat diet (HFD)‐induced lipotoxicity and inflammatory responses occurring in the liver, aorta, and brain.MethodsThe standardized KRG powder was prepared by Korea Ginseng Corporation and was dissolved in phosphate buffered saline as a concentration of 30 mg/mL. Male C57BL/6 mice were fed HFD with or without KRG (10 or 50 mg/kg body weight; orally) for 12 weeks (n = 10 per group). The anti‐inflammatory potential of KRG was determined in the blood and the aorta, liver, and brain.ResultsKRG significantly inhibited HMG‐CoA reductase activity in vitro. In vivo, KRG supplementation suppressed HFD‐associated body weight gain, lipid profile changes, excessive fat deposition in the liver, and increases of leptin, insulin, and ALT levels in the blood. Inflammatory markers of the aorta (ICAM‐1 protein level and wall thickening of the aorta), liver (COX‐2 expression and liver function tests), and brain (APP, BACE1, and Tau protein levels) were also significantly reduced by KRG. In microvascular endothelial cells, 15% cyclic stretch‐mediated upregulation of ICAM‐1 and VCAM‐1 expression was significantly attenuated in the presence of KRG.ConclusionKRG supplementation attenuates HFD‐mediated body weight gain, lipid profile changes, and multi‐tissue inflammatory responses including the liver, aorta, and brain.

Inhibitory effects of curcumin on H2O2-induced cell damage and APP expression and processing in SH-SY5Y cells transfected with APP gene with Swedish mutation.

Alzheimer's disease (AD) is a neurodegenerative disorder, and the pathological mechanism of the disease is still far to understand. According to the amyloid cascade hypothesis in AD, Amyloid-β (Aβ) is considered as a key substance that contributes AD development. Aβ is a β-cleaving product from Amyloid-β protein precursor (APP). Mutations of APP including APPKM670/671670NL (Swedish mutation) result in Aβ overproduction and the development of early-onset familial AD. Increase of oxidative stress and damage also occurs in early stage of AD. In this study, we used a SH-SY5Y cell line that stably expresses APP gene with Swedish mutation (SH-SY5Y-APPswe), and the inhibitory effects of curcumin on H2O2-induced cell damage and APP processing were investigated. Cells were treated with curcumin (0 ~ 5μM) for 4h before hydrogen peroxide (H2O2). Cell growth was detected with CCK-8 assay, and cell damage was determined through the evaluation of release of lactate dehydrogenase (LDH) from the cytosol to the culture medium and the morphological change of nucleus. The ability of mitochondrial stress and the depolarization of mitochondrial membrane potential were assayed through the measuring the oxygen consumption rate (OCR) and the green/red fluorescence ratio of JC-1 dye respectively. The protein levels of APP, sAPPα, sAPPβ, and BACE1 were analyzed with Western blot assay. Aβ production was measured with enzyme-linked immunosorbent assay (ELISA). The results indicated that curcumin inhibits H2O2-induced decrease of cell growth and cell damage. Curcumin attenuates H2O2-induced damage on the ability to mitochondrial oxidative phosphorylation and membrane potential. Curcumin inhibits H2O2-induced increase of APP cleavage through β-cleavage pathway and of intracellular Aβ production. These results imply that curcumin can be used to treat AD through inhibiting oxidative damage-induced APP β-cleavage and intracellular Aβ generation.

Disruption of synaptic expression pattern and age-related DNA oxidation in a neuronal model of lead-induced toxicity

Lead (Pb) is recognized as a potent inducer of synaptic toxicity generally associated with reduced synaptic transmission and increased neuronal fiber excitability, becoming an environmental risk for neurodegenerative processes. Despite numerous toxicological studies on Pb have been directed to the developing brain, attention concerning long-term consequences of pubertal chronic Pb exposure on neuronal activity is still lacking. Thus, we exposed 4-week-old male mice to 0.2 % lead acetate solution for one month, then, conducted behavioral tests or extracted brain homogenate from mice prefrontal cortex (PFC) and hippocampus at the age of 4, 13 and 16-month-old respectively. Our results showed that treated mice exhibited an evident increase in latency to reach platform following pubertal Pb exposure and aging. The increase of 8-OHdG revealed evident neural DNA oxidative damage across time upon pubertal Pb exposure. In the hippocampus of lead exposed mice at three age nodes, the expression of brain-derived neurotrophic factor precursor (proBDNF) increased, while that of mature BDNF (mBDNF), cAMP-response element binding protein (CREB) and phosphorylated CREB (pCREB) decreased compared with the control group. Furthermore, the expression of BACE1 protein and tau phosphorylation level in PFC and hippocampus increased, APP mRNAs in PFC and prolonged induction of BACE1 in hippocampus. Our results show that chronic Pb exposure from pubertal stage onward can either initiate divergent synaptic-related gene expression patterns in adulthood or trigger time-course of neurodegenerative profile within the PFC or hippocampus, which can contribute consistent deficits of cognition across subsequent age-nodes.

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.

Bazhu Decoction, a Traditional Chinese Medical Formula, Ameliorates Cognitive Deficits in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder associated with aging. There are currently no effective treatments for AD. Bazhu decoction (BZD), a traditional Chinese medicine (TCM) formula, has been employed clinically to alleviate AD. However, the underlying molecular mechanisms are still unclear. Here we found that middle- and high-doses of BZD ameliorated the behavioral aspects of 5xFAD transgenic mice in elevated plus maze, Y maze and Morris water maze tests. Moreover, BZD reduced the protein levels of BACE1 and PS1, resulting in a reduction of Aβ plaques. We also identified a beneficial effect of BZD on oxidative stress by attenuating MDA levels and SOD activity in the brains of 5xFAD mice. Together, these results indicate that BZD produces a dose-dependent positive effect on 5xFAD transgenic mouse model by decreasing APP processing and Aβ plaques, and by ameliorating oxidative damage. BZD may play a protective role in the cognitive and anxiety impairments and may be a complementary therapeutic option for 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.

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.

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.

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.

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.

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.

Biliverdin reductase-A impairment links brain insulin resistance with increased Aβ production in an animal model of aging: Implications for Alzheimer disease

Brain insulin resistance is associated with an increased Aβ production in AD although the molecular mechanisms underlying this link are still largely unknown. Biliverdin reductase-A (BVR-A) is a unique Ser/Thr/Tyr kinase regulating insulin signalling. Studies from our group, demonstrated that BVR-A impairment is among the earliest events favoring brain insulin resistance development. Furthermore, reported a negative association between BVR-A protein levels/activation and BACE1 protein levels in the parietal cortex of aged beagles (an animal model of AD), thus suggesting a possible interaction. Therefore, we aimed to demonstrate that BVR-A impairment is a molecular bridge linking brain insulin resistance with increased Aβ production. Age-associated changes of BVR-A, BACE1, insulin signalling cascade and APP processing were evaluated in the parietal cortex of beagles and experiments to confirm the hypothesized mechanism(s) have been performed in vitro in HEK293APPswe cells. Our results show that BVR-A impairment occurs early with age and is associated with brain insulin resistance. Furthermore, we demonstrate that BVR-A impairment favors CK1-mediated Ser phosphorylation of BACE1 (known to mediate BACE1 recycling to plasma membrane) along with increased Aβ production in the parietal cortex, with age. Overall, our results suggest that the impairment of BVR-A is an early molecular event contributing to both (I) the onset of brain insulin resistance and (II) the increased Aβ production observed in AD. We, therefore, suggest that by targeting BVR-A activity it could be possible to delay the onset of brain insulin resistance along with an improved regulation of the APP processing.

Evidence of Increased Prefrontal Cortex Inflammation and Amyloid Precursor Protein Processing in a Translational Swine Model of Heart Failure with Preserved Ejection Fraction

The oligomerization of amyloid beta peptides (Aβ), a histopathological hallmark of diseases such as Alzheimer's, may be elevated in cardiogenic dementias coincident with heart failure (HF). BACE1, the rate‐limiting protease enzyme in the amyloidogenic process, initiates the processing of amyloid precursor protein (APP) leading to the production of Aβ. Understanding the mechanistic regulation of BACE1 may be critical for improving treatment efficacy in heart failure patients exhibiting cardiogenic dementia. The purpose of this study was to examine the molecular mechanisms of cardiogenic dementia in a novel swine model of HF with preserved ejection fraction (HFpEF) exhibiting comorbidities including obesity and metabolic syndrome. Female Ossabaw pigs were randomly divided into two groups; control (CON, n=4) and Western diet‐induced obesity/metabolic syndrome with pressure overload‐induced HFpEF (HF, n=4). The HF group was placed on a Western diet (starting at 3 mo. of age) and aortic‐banded (at 6 mo. of age) prior to euthanasia (at 12 mo. old). At euthanasia, prefrontal cortex and hippocampus regions were collected for Western blot analysis. Total body mass was increased (CON=42±2 vs. HF=72±4 Kg; p<0.01), but brain mass was decreased (CON=110±3 vs. HF=92±2 g; p<0.01) in the HF group. The HF group exhibited greater prefrontal cortex total APP (p<0.01), BACE1 (p<0.01) and CTF protein levels (p=0.01), indicative of increased APP processing. This was accompanied by increased markers of chronic stress and neuroinflammation, specifically phosphorylation of JNK (p<0.01) and phosphorylation of ERK (p<0.01). Aberrant control of the insulin‐signaling pathway was also observed in the prefrontal cortex via increases in the phosphorylation of Akt serine 473 (p<0.01) and insulin‐degrading enzyme protein content (p=0.03) in the HF group. Similar results were observed in the hippocampus for increases in APP processing (increased APP and CTF content), neuroinflammation (phosphorylation of JNK and ERK). However, there were no changes in BACE1 content or the insulin‐signaling pathway, highlighting the importance of examining regional differences in the brain. These data reveal that inflammatory stress and aberrant control of insulin signaling may be implicated in the pathogenic regulation of amyloid beta peptide metabolism in an experimental, large animal setting of HFpEF, and potentially contribute to the cardiogenic dementia often observed in HF patients.Support or Funding InformationNIH R01 HL112998, PI: CAENIH K01 HL125503 – PI, PadillaAmerican Heart Association #16POST2776005, PI: TDOUniversity of Missouri Research Board – PI's, Emter and RectorAlzheimer Society of Brant, Haldimand, Norfolk, Hamilton, and Halton, PI: REKMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sarsasapogenin suppresses Aβ overproduction induced by high glucose in HT-22 cells

The aim of this study is to investigate effects and potential mechanisms of sarsasapogenin (Sar), an active component purified from Rhizoma Anemarrhenae, on high glucose-induced amyloid-beta (Aβ) peptide overproduction in HT-22 cells. HT-22 cells were divided into normal glucose; high glucose (HG); HG co-treated with low, middle, and high concentration of Sar (1, 5, 25μmol/L); and peroxisome proliferator-activated receptor γ (PPARγ) agonist (10μmol/L pioglitazone). After treatment for 24h, protein expression of Aβ and β-site Aβ precursor protein cleaving enzyme 1 (BACE1) and activated PPARγ level were determined by Western blot; Aβ42 levels were also measured by using both immunofluorescence and ELISA methods. BACE1 activity and mRNA level were assessed by fluorospectrophotometry and quantitative PCR, respectively. Cell viability was assayed with a CCK-8 kit. Elevated Aβ expression and Aβ42 level were found in HG-treated HT-22 cells, accompanied by increased BACE1 protein and mRNA levels as well as enzymatic activity, which was markedly attenuated by three concentrations of Sar and pioglitazone. Moreover, HG reduced nuclear PPARγ levels, which was reversed by middle and high concentrations of Sar as well as pioglitazone. PPARγ antagonist GW9662 (20μmol/L) pretreatment reversed the effect of Sar on BACE1 protein expression in HG-cultured HT-22 cells. Additionally, Sar suppressed HG-induced decreases in cell viability of HT-22 cells. High glucose can induce an increase in Aβ levels and a decrease in cell viability in HT-22 cells, while co-treatment with Sar improves these results, which is mediated likely through activation of PPARγ and subsequent downregulation of BACE1.

Vitamin D and Its Analogues Decrease Amyloid-β (Aβ) Formation and Increase Aβ-Degradation.

Alzheimer’s disease (AD) is characterized by extracellular plaques in the brain, mainly consisting of amyloid-β (Aβ), as derived from sequential cleavage of the amyloid precursor protein. Epidemiological studies suggest a tight link between hypovitaminosis of the secosteroid vitamin D and AD. Besides decreased vitamin D level in AD patients, an effect of vitamin D on Aβ-homeostasis is discussed. However, the exact underlying mechanisms remain to be elucidated and nothing is known about the potential effect of vitamin D analogues. Here we systematically investigate the effect of vitamin D and therapeutically used analogues (maxacalcitol, calcipotriol, alfacalcidol, paricalcitol, doxercalciferol) on AD-relevant mechanisms. D2 and D3 analogues decreased Aβ-production and increased Aβ-degradation in neuroblastoma cells or vitamin D deficient mouse brains. Effects were mediated by affecting the Aβ-producing enzymes BACE1 and γ-secretase. A reduced secretase activity was accompanied by a decreased BACE1 protein level and nicastrin expression, an essential component of the γ-secretase. Vitamin D and analogues decreased β-secretase activity, not only in mouse brains with mild vitamin D hypovitaminosis, but also in non-deficient mouse brains. Our results further strengthen the link between AD and vitamin D, suggesting that supplementation of vitamin D or vitamin D analogues might have beneficial effects in AD prevention.

The Decrease of Uch-L1 Activity Is a Common Mechanism Responsible for Aβ 42 Accumulation in Alzheimer's and Vascular Disease.

Alzheimer’s disease (AD) is a multifactorial pathology causing common brain spectrum disorders in affected patients. These mixed neurological disorders not only include structural AD brain changes but also cerebrovascular lesions. The main aim of the present issue is to find the factors shared by the two pathologies. The decrease of ubiquitin C-terminal hydrolase L1 (Uch-L1), a major neuronal enzyme involved in the elimination of misfolded proteins, was observed in ischemic injury as well as in AD, but its role in the pathogenesis of AD is far to be clear. In this study we demonstrated that Uch-L1 inhibition induces BACE1 up-regulation and increases neuronal and apoptotic cell death in control as well as in transgenic AD mouse model subjected to Bengal Rose, a light-sensitive dye inducing that induces a cortical infarction through photo-activation. Under the same conditions we also found a significant activation of NF-κB. Thus, the restoration of Uch-L1 was able to completely prevent both the increase in BACE1 protein levels and the amount of cell death. Our data suggest that the Uch-L1-mediated BACE1 up-regulation could be an important mechanism responsible for Aβ peptides accumulation in vascular injury and indicate that the modulation of the activity of this enzyme could provide new therapeutic strategies in AD.

Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.