Β-site Amyloid Precursor Protein Cleavage Research Articles

Application of Dominant Gut Microbiota Promises to Replace Fecal Microbiota Transplantation as a New Treatment for Alzheimer's Disease.

Several studies have confirmed that the pathophysiological progression of Alzheimer's disease (AD) is closely related to changes in the intestinal microbiota; thus, modifying the intestinal microbiota has emerged as a new way to treat AD. Effective interventions for gut microbiota include the application of probiotics and other measures such as fecal microbiota transplantation (FMT). However, the application of probiotics ignores that the intestine is a complete microecosystem with competition among microorganisms. FMT also has issues when applied to patient treatment. In a previous study, we found that eight species of bacteria that are isolated with high frequency in the normal intestinal microbiota (i.e., intestinal dominant microbiota) have biological activities consistent with the effects of FMT. In this article, we confirmed that the treatment of intestinal dominant microbiota significantly restored intestinal microbiota abundance and composition to normal levels in APP/PS1 mice; downregulated brain tissue pro-inflammatory cytokines (IL-1β and IL-6) and amyloid precursor protein (APP) and β-site APP cleavage enzyme 1 (BACE1) expression levels; and reduced the area of Aβ plaque deposition in the brain hippocampus. Our study provides a new therapeutic concept for the treatment of AD, adjusting the intestinal microecological balance through dominant intestinal microbiota may be an alternative to FMT.

Antioxidant and anti-Alzheimer activities of Clivia miniata (Lindl) roots, bulbs, and aerial parts: In-vitro and in-silico studies

Clivia miniata (Lindl) is a member of the family Amaryllidaceae known for its chemically diverse alkaloids with a wide range of biological activities. Many reports revealed a direct role of oxidative stress in the early stage of Alzheimer’s disease (AD). Meanwhile, β-site amyloid precursor protein cleavage enzyme 1 (BACE-1) is a molecular target for the treatment of AD. We aimed to investigate C. miniata root, bulb, and aerial part chemical profiling, antioxidant, BACE-1, and AChE enzyme inhibitory activities. Results showed that the total root had the most potent radical scavenging activity as compared to the total bulb and aerial part, respectively. Ethanol root extract had the most potent BACE-1 inhibitory activity (IC50 = 0.02 ± 0.001 µg/mL) as compared to the bulb and aerial part (IC50 = 0.93 ± 0.13, 1.80 ± 0.24 µg/mL), respectively. Moreover, the total root extract mitigated AChE enzyme activity more than total bulb and aerial fractions with IC50 values of (0.06 ± 0.02, 0.58 ± 0.3, and 1.89 ± 0.42 µg/mL, respectively. Bioassay-guided acid-base fractionation confirmed superior BACE-1 inhibitory activity of the root fractions particularly, methylene chloride and ethyl acetate fractions with (IC50 values of 0.21 ± 0.60 and 0.01 ± 0.001 µg/mL), respectively. UPLC-MS analysis of ethyl acetate and methylene chloride fractions of C. miniata root led to the identification of eight phenolics and thirteen alkaloids, respectively. Molecular docking studies against BACE-1 protein revealed that lycorine di-hexoside, miniatine, and cliviaaline were the most promising hits. Further investigation of anti-AD potential of the aforementioned small molecules is required.

5-Methyltetrahydrofolate Alleviates Memory Impairment in a Rat Model of Alzheimer's Disease Induced by D-Galactose and Aluminum Chloride.

The effects of 5-methyltetrahydrofolate (5-MTHF) on a rat model of Alzheimer's disease (AD) induced by D-galactose (D-gal) and aluminum chloride (AlCl3) were investigated. Wistar rats were given an i.p. injection of 60 mg/kg D-gal and 10 mg/kg AlCl3 to induce AD and three doses of 1 mg/kg, 5 mg/kg or 10 mg/kg 5-MTHF by oral gavage. A positive control group was treated with 1 mg/kg donepezil by gavage. Morris water maze performance showed that 5 and 10 mg/kg 5-MTHF significantly decreased escape latency and increased the number of platform crossings and time spent in the target quadrant for AD rats. The administration of 10 mg/kg 5-MTHF decreased the brain content of amyloid β-protein 1-42 (Aβ1-42) and phosphorylated Tau protein (p-Tau) and decreased acetylcholinesterase and nitric oxide synthase activities. Superoxide dismutase activity, vascular endothelial growth factor level and glutamate concentration were increased, and malondialdehyde, endothelin-1, interleukin-6, tumor necrosis factor-alpha and nitric oxide decreased. The administration of 10 mg/kg 5-MTHF also increased the expression of disintegrin and metallopeptidase domain 10 mRNA and decreased the expression of β-site amyloid precursor protein cleavage enzyme 1 mRNA. In summary, 5-MTHF alleviates memory impairment in a D-gal- and AlCl3-exposed rat model of AD. The inhibition of Aβ1-42 and p-Tau release, reduced oxidative stress, the regulation of amyloid precursor protein processing and the release of excitatory amino acids and cytokines may be responsible.

Preventive effects of arctigenin from Arctium lappa L against LPS-induced neuroinflammation and cognitive impairments in mice.

Arctigenin (Arc) is a phenylpropanoid dibenzylbutyrolactone lignan in Arctium lappa L, which has been widely applied as a traditional Chinese herbal medicine for treating inflammation. In the present study, we explored the neuroprotective effect and the potential mechanisms of arctigenin against LPS-evoked neuroinflammation, neurodegeneration, and memory impairments in the mice hippocampus. Daily administration of arctigenin (50mg/kg per day, i.g.) for 28days revealed noticeable improvements in spatial learning and memory deficits after exposure to LPS treatment. Arctigenin prevented LPS-induced neuronal/synaptic injury and inhibited the increases in Abeta (Aβ) generation and the levels of amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1). Moreover, arctigenin treatment also suppressed glial activation and reduced the production of proinflammatory cytokines. In LPS-treated BV-2 microglial cells and mice, activation of the TLR4 mediated NF-κB signaling pathway was significantly suppressed by arctigenin administration. Mechanistically, arctigenin reduced the LPS-induced interaction of adiponectin receptor 1 (AdipoR1) with TLR4 and its coreceptor CD14 and inhibited the TLR4-mediated downstream inflammatory response. The outcomes of the current study indicate that arctigenin mitigates LPS-induced apoptotic neurodegeneration, amyloidogenesis and neuroinflammation as well as cognitive impairments, and suggest that arctigenin may be a potential therapeutic candidate for neuroinflammation/neurodegeneration-related diseases.

Extracellular-Signal-Regulated Kinase Inhibition Switches APP Processing from β- to α-Secretase under Oxidative Stress: Modulation of ADAM10 by SIRT1/NF-κB Signaling.

The sequential cleavage of full-length amyloid precursor protein (APP) by secretases has been at the center of efforts for understanding the onset of Alzheimer's disease (AD). A decrease in α-secretase activity was observed during the progression of AD; however, the precise molecular mechanism involved in the downregulation of α-secretase under oxidative stress is not fully understood. In the present study, we have demonstrated that pharmacological inhibition of mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) by mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor (PD98059) restored the expression of a disintegrin and metalloproteinase 10 (ADAM10) with a concomitant decrease in β-site APP cleavage enzyme 1 (BACE1) under oxidative stress. Silent mating-type information regulation 2 homologue 1 (SIRT1) activation by resveratrol also mitigated alterations in secretase levels through MAPK/ERK signaling. Intracerebroventricular (ICV) administration of streptozotocin in rats showed amyloidogenic processing of APP and altered the SIRT1/ERK axis in the hippocampus. We also observed that the ADAM10 expression is controlled at the transcriptional level by oxidative stress. Using the luciferase reporter activity of ADAM10 promoter deletion constructs, we have identified the region 290 bp upstream of the transcription start site (TSS) possessing regulatory elements responsible for ADAM10 downregulation with hydrogen peroxide (H2O2) treatment. Further, bioinformatics analysis revealed the presence of putative nuclear factor kappa B (NF-κB) binding sites in the ADAM10 promoter region. Treatment of cortical neurons with the NF-κB inhibitor (Bay 11-7082) mitigated the transcriptional upregulation of ADAM10 by PD98059. Overall, our findings suggest that SIRT1/ERK/NF-κB axis contributes to the downregulation of ADAM10, resulting in the shift from nonamyloidogenic to amyloidogenic processing of APP under oxidative stress.

Vitamin D deficiency exacerbates Alzheimer-like pathologies by reducing antioxidant capacity

Vitamin D (VD) deficiency is prevalent among aging people and Alzheimer's disease (AD) patients. However, the roles of VD deficiency in the pathology of AD remain largely unexplored. In this study, APP/PS1 mice were fed a VD-deficient diet for 13 weeks to evaluate the effects of VD deficiency on the learning and memory functions and the neuropathological characteristics of the mice. Our study revealed that VD deficiency accelerated cognitive impairment in the APP/PS1 mice. Mechanistic studies revealed that VD deficiency promoted glial activation and increased inflammatory factor secretion. Furthermore, VD deficiency increased the production and deposition of Aβ by elevating the expression levels of amyloid precursor protein (APP) and β-site APP cleavage enzyme 1 (BACE1). In addition, VD deficiency increased the phosphorylation of Tau at Thr181, Thr205 and Ser396 by increasing the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3α/β (GSK3α/β) and promoted synaptic dystrophy and neuronal loss. All these effects of VD deficiency may be ascribed to enhanced oxidative stress via the downregulation of superoxide dismutase 1 (SOD1), glutathione peroxidase 4 (GPx4) and cystine/glutamate exchanger (xCT). Taken together, our data suggest that VD deficiency exacerbates Alzheimer-like pathologies via promoting inflammatory stress, increasing Aβ production and elevating Tau phosphorylation by decreasing antioxidant capacity in the brains of APP/PS1 mice. Hence, rescuing the VD status of AD patients should be taken into consideration during the treatment of AD.

Identification of ovalbumin‐derived peptides as multi‐target inhibitors of AChE, BChE, and BACE1

Alzheimer's disease (AD) is a kind of progressive neurodegenerative disease that affects the elderly. There is no ideal treatment for AD. Thus, the purpose of this study is to identify anti-AD peptides from ovalbumin. The potential tripeptides IEK, LYR, and CIK were selected for molecular docking. The '-CDOCKER_Energy' values of the best docking positions of the tripeptide IEK, LYR, and CIK interacting with acetylcholinesterase (AChE) were 93.8119, 86.9556 and 73.6370 kcal mol-1 , respectively. The '-CDOCKER_Energy' values for interaction with butyrylcholinesterase (BChE) were 96.6386, 80.8392, and 87.4341 kcal mol-1 , respectively. Most importantly, the '-CDOCKER_Energy' values for interaction with β-site amyloid precursor protein cleavage enzyme1 (BACE1) were 85.5903, 71.3342, and 68.4290 kcal mol-1 , respectively. Overall, in vitro assay results demonstrated that the peptide CIK exhibited impressive inhibitory activities against AChE, BChE, and BACE1, with half maximal inhibitory concentration (IC50 ) values of 6.76, 7.72, and 34.48 μmol L-1 , respectively. In particular, CIK can be joined with some peripheral anion sites (PAS) and catalytic sites on AChE, BChE, and BACE1. Tripeptide CIK can effectively inhibit the activities of AChE, BChE, and BACE1. Tripeptide CIK therefore has the potential to treat AD effectively. © 2020 Society of Chemical Industry.

Geraniin Attenuates Lipopolysaccharide-Induced Cognitive Impairment in Mice by Inhibiting Toll-Like Receptor 4 Activation.

Geraniin has been reported to possess potent anti-inflammatory properties and to modulate the macrophage polarization. This study sought to evaluate the protective effects and underlying mechanisms of geraniin on lipopolysaccharide (LPS)-induced neuroinflammation and neurobiological alternations as well as cognitive impairment. Daily intragastrical administration with geraniin (20 mg kg-1 day-1) for 14 days significantly prolonged the duration in the target quadrant (26.53 ± 2.03 versus 37.09 ± 3.27%; p < 0.05) and increased crossing-target number (1.93 ± 0.22 versus 3.08 ± 0.17; p < 0.01) in the probe test of LPS-treated mice. Geraniin also ameliorated LPS-elicited neural/synaptic impairments and decreased levels of LPS-induced Aβ generation (p < 0.05), amyloid precursor protein (APP) (p < 0.05) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1) (p < 0.05). Furthermore, geraniin suppressed the production of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) (9.85 ± 0.58 versus 5.20 ± 0.52 pg/mg of protein; p < 0.01), interleukin (IL)-1β (16.31 ± 0.67 versus 8.62 ± 0.46 pg/mg of protein; p < 0.01), and IL-6 (12.12 ± 0.45 versus 7.43 ± 0.32 pg/mg of protein; p < 0.05), and inhibited glial cell activation. Moreover, geraniin effectively polarized the microglia toward an anti-inflammatory M2 phenotype. Further study revealed that geraniin targeted toll-like receptor 4 (TLR4)-mediated signaling and decreased the production of pro-inflammatory cytokines in BV-2 microglial cells. These results indicate that geraniin mitigates LPS-elicited neural/synaptic neurodegeneration, amyloidogenesis, neuroinflammation, and cognitive impairment and suggest geraniin as a therapeutic option for neuroinflammation-associated neurological disorders, such as Alzheimer's disease.

Pseudoginsenoside-F11 alleviates cognitive deficits and Alzheimer's disease-type pathologies in SAMP8 mice.

Alzheimer's disease (AD) is a common neurodegenerative disease which is characterized by aggregation of amyloid beta (Aβ) and hyperphosphorylated tau. We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, improved cognitive function and reduced Aβ aggregation in APP/PS1 mice, a familial AD model. Here, we chose senescence-accelerated mouse prone 8 (SAMP8) mice, a widely used model of aging, to investigate the effect of PF11 on sporadic AD. PF11 was orally administered to male 6-month-old SAMP8 mice for 3 months. Consistent with previous studies, SAMP8 mice showed several AD-type pathologies including cognitive impairment, Aβ deposition and tau hyperphosphorylation. We found increased protein levels of cytoplasmic amyloid precursor protein (APP) and β-site APP cleavage enzyme 1 (BACE1) in the hippocampus and cortex of SAMP8 mice. The protein level of demethylated protein phosphatase 2A (PP2A) was elevated in SAMP8 animals and the protein level of leucine carboxyl methyltransferase 1 (LCMT-1) was reduced. PF11 attenuated learning and memory impairments in the novel object recognition test and Morris water maze. PF11 promoted the transport of APP from cytoplasm to plasma membrane and decreased the abnormally high expression of BACE1 in hippocampus and cortex of SAMP8 mice. The elevated protein level of demethylated PP2A and the reduced expression of LCMT-1 in hippocampus and cortex of SAMP8 were also attenuated by PF11. Together, our findings indicate that PF11 has beneficial effects on AD-like pathological changes in SAMP8 mice and may act by inhibiting amyloidogenic processing of APP and attenuating tau hyperphosphorylation.

Icariside II Ameliorates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Inhibiting the Amyloidogenic Pathway: Involvement of BDNF/TrkB/CREB Signaling and Up-Regulation of PPARα and PPARγ in Rats.

Chronic cerebral hypoperfusion (CCH) is regarded as a high-risk factor for cognitive decline of vascular dementia (VD) as it is conducive to induce beta-amyloid (Aβ) aggregation. Icariside II (ICS II), a plant-derived flavonoid compound, has showed neuroprotective effect on animal models of Alzheimer’s disease (AD) by decreasing Aβ levels. Here, we assessed the effect of ICS II on CCH-induced cognitive deficits and Aβ levels in rats, and the possible underlying mechanisms were also explored. It was disclosed that CCH induced by bilateral common carotid artery occlusion (BCCAO) caused cognitive deficits, neuronal injury and increase of Aβ1-40 and Aβ1-42 levels in the rat hippocampus, while oral administration of ICS II for 28 days abolished the above deficits in the hippocampus of BCCAO rats. Meanwhile, ICS II significantly decreased the expression of beta-amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1), as well as increased the expression of a disintegrin and metalloproteinase domain 10 (ADAM10) and insulin-degrading enzyme (IDE). ICS II also activated peroxisome proliferator-activated receptor (PPAR)α and PPARγ, enhanced the expression of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB), levels of Akt and cAMP response element binding protein (CREB) phosphorylation. Together, these findings suggested that ICS II attenuates CCH-induced cognitive deficits by inhibiting the amyloidogenic pathway via involvement of BDNF/TrkB/CREB signaling and up-regulation of PPARα and PPARγ in rats.

Honokiol Ameliorates Amyloidosis and Neuroinflammation and Improves Cognitive Impairment in Alzheimer's Disease Transgenic Mice.

The present study examined the effects of honokiol on amyloid-β (Aβ)-induced cognitive impairment and the underlying mechanisms in APPswe/PS1dE9 transgenic mice. The results showed that honokiol administration (20 mg/kg per day, intraperitoneally) for 6 weeks effectively improved spatial memory deficits in APPswe/PS1dE9 transgenic mice. Honokiol significantly lowered Aβ production and senile plaque deposition by downregulating β-site amyloid precursor protein cleavage enzyme 1 and enhancing Aβ phagocytosis by microglia. Honokiol reduced glial cell activation and the production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Honokiol increased the transcriptional activity and protein levels of peroxisome proliferator-activated receptor-γ (PPARγ) However, all of the beneficial effects of honokiol on pathologic changes, including biochemistry and cognitive function, could be blocked by GW9662, a specific PPARγ inhibitor. These findings suggested that honokiol may be a natural PPARγ agonist, acting to attenuate Aβ generation and neuroinflammation. Therefore, honokiol may be a potential therapeutic approach for Alzheimer's disease.

Flavonoid-rich ethanol extract from the leaves of Diospyros kaki attenuates cognitive deficits, amyloid-beta production, oxidative stress, and neuroinflammation in APP/PS1 transgenic mice

Amyloid-β peptide (Aβ) initiates a cascade of pathological events, including activation of microglial cells, oxidative stress, and inflammation, leading to neuronal death and the typical pathological changes in Alzheimer’s disease (AD). Flavonoids have been reported to exert neuroprotective activities, not only through their generally accepted antioxidant effects, but also through their ability to protect against neurotoxin-induced injury. Flavonoids reduce Aβ production, inhibit neuroinflammation, increase cerebrovascular function, and improve cognitive performance. Here, we analyzed the effects of a flavonoid-rich ethanol extract from the leaves of Diospyros kaki (FLDK) in APP/PS1 transgenic mice. We found that oral treatment with FLDK reversed learning and memory impairment, reduced Aβ burden and expression of β-site amyloid precursor protein cleavage enzyme 1 (BACE1), and decreased microglial activation in senile plaques. FLDK restored antioxidant enzyme activities, as well as reduced the lipid peroxidation product, malondialdehyde, and inflammatory mediators. These results demonstrate that FLDK alleviates cognitive decline and reduces Aβ burden, microglial activation, oxidative stress, and inflammation responses. Thus, FLDK treatment may be a potential therapeutic strategy for preventing and treating AD, at least in part via its anti-oxidant and anti-inflammatory biological activities and its effect on the Aβ producing enzyme BACE1.

Isoflurane anesthesia promotes cognitive impairment by inducing expression of β-amyloid protein-related factors in the hippocampus of aged rats.

Isoflurane anesthesia has been shown to be responsible for cognitive impairment in Alzheimer’s disease (AD) and development of AD in the older age groups. However, the pathogenesis of AD-related cognitive impairments induced by isoflurane anesthesia remains elusive. Thus, this study was designed to investigate the mechanism by which isoflurane anesthesia caused AD-related cognitive impairments. Aged Wistar rats were randomly divided into 6 groups (n = 12), 1 control group (CONT) and 5 isoflurane treated (ISO) groups (ISO 0, ISO 0.5D, ISO 1D, ISO 3D and ISO 7D). The CONT group inhaled 30% O2 for 2 h without any anesthesia. ISO groups were placed under anesthesia with 3% isoflurane and then exposed to 1.5% isoflurane delivered in 30% O2 for 2 h. Rats in each ISO group were then analyzed immediately (ISO 0) or at various time points (0.5, 1, 3 or 7 day) after this exposure. Cognitive function was assessed using the Morris water maze test. Protein levels of amyloid precursor protein (APP), β-site APP cleavage enzyme-1 (BACE-1) and Aβ42 peptide were analyzed in hippocampal samples by Western blot. β-Amyloid (Abeta) plaques were detected in hippocampal sections by Congo red staining. Compared with controls, all ISO groups showed increased escape latency and impaired spatial memory. Isoflurane increased APP mRNA expression and APP protein depletion, promoting Aβ42 overproduction, oligomerization and accumulation. However, isoflurane did not affect BACE-1 expression. Abeta plaques were observed only in those ISO groups sacrificed at 3 or 7 d. Our data indicate that aged rats exposed to isoflurane had increased APP mRNA expression and APP protein depletion, with Aβ42 peptide overproduction and oligomerization, resulting in formation of Abeta plaques in the hippocampus. Such effects might have contributed to cognitive impairments, including in spatial memory, observed in these rats after isoflurane anesthesia.

Icariside II Effectively Reduces Spatial Learning and Memory Impairments in Alzheimer's Disease Model Mice Targeting Beta-Amyloid Production.

Icariside II (ICS II) is a broad-spectrum anti-cancer natural compound extracted from Herba Epimedii Maxim. Recently, the role of ICS II has been investigated in central nervous system, especially have a neuroprotective effect in Alzheimer’s disease (AD). In this study, we attempted to investigate the effects of ICS II, on cognitive deficits and beta-amyloid (Aβ) production in APPswe/PS1dE9 (APP/PS1) double transgenic mice. It was found that chronic ICS II administrated not only effectively ameliorated cognitive function deficits, but also inhibited neuronal degeneration and reduced the formation of plaque burden. ICS II significantly suppressed Aβ production via promoting non-amyloidogenic APP cleavage process by up-regulating a disintegrin and metalloproteinase domain 10 (ADAM10) expression, inhibited amyloidogenic APP processing pathway by down-regulating amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1) expression in APP/PS1 transgenic mice. Meanwhile, ICS II attenuated peroxisome proliferator-activated receptor-γ (PPARγ) degradation as well as inhibition of eukaryotic initiation factor α phosphorylation (p-eIF2α) and PKR endoplasmic reticulum regulating kinase phosphorylation (p-PERK). Moreover, phosphodiesterase type 5 inhibitors (PDE5-Is) have recently emerged as a possible therapeutic target for cognitive enhancement via inhibiting Aβ levels, and we also found that ICS II markedly decreased phosphodiesterase-5A (PDE5A) expression. In conclusion, the present study demonstrates that ICS II could attenuate spatial learning and memory impairments in APP/PS1 transgenic mice. This protection appears to be due to the increased ADAM10 expression and decreased expression of both APP and BACE1, resulting in inhibition of Aβ production in the hippocampus and cortex. Inhibition of PPARγ degradation and PERK/eIF2α phosphorylation are involved in the course, therefore suggesting that ICS II might be a promising potential compound for the treatment of AD.

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).

Arctigenin Effectively Ameliorates Memory Impairment in Alzheimer's Disease Model Mice Targeting Both -Amyloid Production and Clearance

Alzheimer's disease (AD) chiefly characterizes a progressively neurodegenerative disorder of the brain, and eventually leads to irreversible loss of intellectual abilities. The β-amyloid (Aβ)-induced neurodegeneration is believed to be the main pathological mechanism of AD, and Aβ production inhibition or its clearance promotion is one of the promising therapeutic strategies for anti-AD research. Here, we report that the natural product arctigenin from Arctium lappa (L.) can both inhibit Aβ production by suppressing β-site amyloid precursor protein cleavage enzyme 1 expression and promote Aβ clearance by enhancing autophagy through AKT/mTOR signaling inhibition and AMPK/Raptor pathway activation as investigated in cells and APP/PS1 transgenic AD model mice. Moreover, the results showing that treatment of arctigenin in mice highly decreased Aβ formation and senile plaques and efficiently ameliorated AD mouse memory impairment strongly highlight the potential of arctigenin in anti-AD drug discovery.

Modest reductions in BACE1 activity significantly reduce beta-amyloid plaque load and neuroinflammation in a mouse model of Alzheimer's disease

Inhibiting the amyloidogenic processing of the amyloid precursor protein (APP) by blocking the activity of the β-site APP cleavage enzyme (BACE) has been extensively pursued as a therapeutic strategy for Alzheimer's disease (AD). Indeed, brief treatment with high doses of anti-BACE1 antibodies effectively reduced peripheral and central Aβ production in wild-type mice and nonhuman primates (Atwal et al., STM 2011), suggesting a potential therapeutic role for this antibody. However, it is still unclear what level of Aβ reduction could translate to a therapeutically meaningful impact on the progression of disease pathology if treatment were continued chronically. Therefore, to test the effectiveness of the anti-BACE1 antibodies for reducing/preventing A β plaque deposition and associated pathology, we treated transgenic mice expressing London mutant (V717I) human APP (hAPPlon) for four months starting at an age prior to the onset of plaque deposition. Female transgenic hAPPlon mice or non-transgenic littermate controls received either short-term treatment (3 IP injections, each separated by 4 days), with control antibody or anti-BACE1 antibodies (30mg/kg, 100mg/kg) starting at 2–3 months-of-age, or chronic treatment (weekly IP injections for 16 weeks) of a control antibody or anti-BACE1 antibody (100mg/kg), starting at 10.5 months-of-age. Serum, plasma and brain tissue was collected for PK/PD and pathology analysis. We found that short-term anti-BACE1 treatment (100mg/kg) modestly reduced guanidine-extracted total brain A β 40 and A β 42 levels by 17% and 31%, respectively, compared with control-treated hAPPlon mice when measured by ELISA. Chronic anti-BACE1 treatment significantly reduced A β plaques by ∼50% throughout the brain, compared with control-treated hAPPlon mice. This was associated with a reduction in total brain A β 40 and A β 42 levels by 73% and 55%, respectively. Plaque-associated neuroinflammation, as measured by microglia and astrocyte activation, was also significantly reduced. Modest but chronic inhibition of BACE1 activity with high affinity anti-BACE1 antibodies can significantly reduce A β plaque deposition and associated pathology in a mouse model of AD. This suggests that partial inhibition of BACE1 could be an effective therapeutic strategy in human AD patients as well.

P1‐249: Role of BACE1 and BACE2 in the production of endogenous mouse Aß

The imbalance between Aβ production and/or clearance is hypothesized to be the major contributor to pathogenesis in Alzheimer's disease (AD). Inhibiting the sequential proteolytic cleavage of amyloid precursor protein (APP) by β- or γ-secretase blockade has been extensively pursued as a therapeutic strategy for AD treatment. The β-site APP cleavage enzyme (BACE) remains a popular target for small molecule inhibitors and antibodies because of its rate-limiting role in Aβ production. Targeting BACE1, versus its close homolog BACE2, is favored because BACE1 is highly expressed in neurons of the brain, whereas BACE2 is not. Although much is known about the role of BACE1 and BACE2 in processing human APP in in vitro and in vivo over-expression systems, less is known about their role in processing endogenous mouse APP in the brain and periphery. To determine the contribution of BACE1 and BACE2 to endogenous Aβ production in the brain and periphery, we measured brain and plasma Aβ40 levels by ELISA from BACE1ko, BACE2ko, and BACE1/2 double ko mice. Endogenous Aβ40 levels were measured in wild-type or BACE1ko mice to determine the selectivity of anti-BACE1 antibodies and small molecules against BACE1. In this study, we demonstrated that BACE1 contributes to ∼90% of endogenous brain and 50-60% of plasma Aβ40 production, whereas BACE2 does not contribute to either brain or plasma Aβ40 production. APPko mice had no detectable Aβ40 levels, demonstrating the specificity of the assay. Transient handling-induced increases in peripheral Aβ40 are mediated by BACE1, not BACE2. Anti-BACE1 antibodies are selective in inhibiting BACE1 activity, since no further reductions in Aβ40 levels were observed in BACE1 ko mice. BACE1 is the major β-secretase involved in the production of endogenous mouse Aβ in the brain and periphery, whereas BACE2 has little to no role. The remaining peripheral Aβ production in BACE1/2 ko mice suggests that there may be additional β-secretases yet to be identified.

Mutant Presenilin 2 Increases β-Secretase Activity Through Reactive Oxygen Species–Dependent Activation of Extracellular Signal–Regulated Kinase

Senile plaques composed of β-amyloid (Aβ) are a pathological hallmark of Alzheimer disease. Presenilin 2 (PS2) mutations increase Aβ generation in the brains of Alzheimer disease patients, but the underlying mechanism of Aβ generation by PS2 mutations remains to be clarified. The Aβ is generated through the sequential cleavage of amyloid precursor protein by β- and γ-secretases. Here, we show that the PS2 mutation N141I enhances the activity of β-secretase and expression of the β-site amyloid precursor protein cleavage enzyme 1, a major neuronal β-secretase in the brains of PS2 transgenic mice and in PC12 cells overexpressing mutant PS2. In parallel with the increased activity of β-secretase, activation of extracellular signal-regulated kinase (ERK), Aβ1-40 and Aβ1-42 levels, generation of reactive oxygen species, and lipid peroxidation were higher in the mutant mouse neurons and the PC12 cells. Colocalization of phosphorylated ERK (phospho-ERK) and β-site amyloid precursor protein cleavage enzyme 1 with hydroxynonenal-histidine was found in the mutant brains. An ERK inhibitor U0126 and an antioxidant N-acetylcysteine prevented the expression and activity of β-secretase, ERK activation, and reactive oxygen species generation in both neurons and PC12 cells expressing mutant PS2 in a dose-dependent manner. Together, these data suggest that oxidative stress-mediated ERK activation contributes to increases in β-secretase and, thus, an increase of Aβ generation in neuronal cells expressing mutant PS2.

Inhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloid β and APP in SK-N-SH Cells

The study was undertaken to explore whether piperlonguminine/dihydropiperlonguminine could inhibit the production of amyloidbeta (Abeta) in human neuroblastoma cells (SK-N-SH) and to examine the underlying mechanism of this effect. Piperlonguminine/dihydropiperlonguminine components (1:0.8) were extracted from Futokadsura stem, and then used to treat SK-N-SH cells at three different concentrations: 3.13 microg/ml, 6.25 microg/ml and 12.50 microg/ml. Subsequently, the production of Abeta42 and Abeta40 were measured by Western blot analysis and enzyme linked immunosorbent assay (ELISA). On the other hand, the expressions of amyloid precursor protein (APP), Notch1 (Notch intracellular domain) and beta-site amyloid precursor protein cleavage enzyme (BACE-1) were also examined by Western blot assay. The activities of beta-secretase and gamma-secretase were detected at the same time. Furthermore, Abeta42 level was detected by immunocytochemistry staining. We demonstrated that the treatment of piperlonguminine/dihydropiperlonguminine could significantly decrease the levels of APP, Abeta42 and Abeta40 peptide in SK-N-SH cells, despite the fact that the activities of beta-secretase and gamma-secretase were not affected significantly. These data suggest that piperlonguminine/dihydropiperlonguminine components could significantly inhibit the level of APP, Abeta42 and Abeta40 peptide without affecting the activity of beta-secretase and gamma-secretase in SK-N-SH cells.