Aβ Degradation Research Articles

A self-destructive nanosweeper that captures and clears amyloid \u03b2-peptides

Cerebral amyloid β-peptide (Aβ) accumulation resulting from an imbalance between Aβ production and clearance is one of the most important causes in the formation of Alzheimer’s disease (AD). In order to preserve the maintenance of Aβ homeostasis and have a notable AD therapy, achieving a method to clear up Aβ plaques becomes an emerging task. Herein, we describe a self-destructive nanosweeper based on multifunctional peptide-polymers that is capable of capturing and clearing Aβ for the effective treatment of AD. The nanosweeper recognize and bind Aβ via co-assembly through hydrogen bonding interactions. The Aβ-loaded nanosweeper enters cells and upregulates autophagy thus promoting the degradation of Aβ. As a result, the nanosweeper decreases the cytotoxicity of Aβ and rescues memory deficits of AD transgenic mice. We believe that this resourceful and synergistic approach has valuable potential as an AD treatment strategy.

Abstract 183: Association of LDL Particles With Cerebral Amyloidosis

Cerebral amyloidosis is a condition in which β-Amyloid (Aβ) proteins are deposited in the cerebral cortex and is a predictor of Alzheimer’s disease (AD). In the Aging Brain Study, we reported an association between LDL cholesterol and cerebral amyloidosis assessed using PET PiB imaging. LDL comprises multiple species of varying size, density and protein composition, including very large LDL-I which is enriched in ApoE and can bind to ApoE receptors. In this study, LDL particle fractions were measured in plasma samples of 58 participants (40 women and 18 men) of the Aging Brain study. Cerebral amyloidosis was assessed using Pittsburgh Compound B index-Positron Emission Tomography (PiB-PET) imaging. LDL subfractions were analyzed by the method of ion mobility. The subjects were divided into three groups based on PiB tertiles. Compared to the first tertile, total plasma cholesterol as well as LDL cholesterol were greater in patients in the second and third PiB index tertiles (p values = 0.05 and 0.03 respectively). Amongst the LDL subfractions, levels of LDL-I as well as very small LDL-IVa particles were significantly greater in the second and third PiB index tertiles and independent of LDL cholesterol levels (p values = 0.03 and 0.04 respectively). A significant inverse association was also observed between LDL-I and hippocampal volumes (r=-0.33, p=0.02). We suggest that LDL-I level may be a mechanistic biomarker for extent of cerebral amyloidosis. Lipoprotein receptors, particularly LRP-1, participate in Aβ clearance from the brain and its hepatic degradation. One potential mechanism for our findings is competition between plasma-derived LDL-I and brain Aβ that may retard Aβ clearance and degradation.

Transplantation of Human Menstrual Blood-Derived Mesenchymal Stem Cells Alleviates Alzheimer's Disease-Like Pathology in APP/PS1 Transgenic Mice.

Extracellular β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) are the pathological hallmarks of Alzheimer’s disease (AD). Mesenchymal stem cells (MSCs) have shown therapeutic efficacy in many neurodegenerative diseases, including AD. Human menstrual blood-derived stem cells (MenSCs) are a novel source of MSCs advantageous for their higher proliferation rate and because they are easy to obtain without ethical concerns. Although MenSCs have exhibited therapeutic efficacy in some diseases, their effects on AD remain elusive. In the present study, we showed that intracerebral transplantation of MenSCs dramatically improved the spatial learning and memory of APP/PS1 mice. In addition, MenSCs significantly ameliorated amyloid plaques and reduced tau hyperphosphorylation in APP/PS1 mice. Remarkably, we also found that intracerebral transplantation of MenSCs markedly increased several Aβ degrading enzymes and modulated a panel of proinflammatory cytokines associated with an altered microglial phenotype, suggesting an Aβ degrading and anti-inflammatory impact of MenSCs in the brains of APP/PS1 mice. In conclusion, these findings suggest that MenSCs are a promising therapeutic candidate for AD.

Neuroprotective Effect of Alpha-Linolenic Acid against Aβ-Mediated Inflammatory Responses in C6 Glial Cell.

Therapeutic approaches for neurodegeneration, such as Alzheimer's disease (AD), have been widely studied. One of the critical hallmarks of AD is accumulation of amyloid beta (Aβ). Aβ induces neurotoxicity and releases inflammatory mediators or cytokines through activation of glial cell, and these pathological features are observed in AD patient's brain. The purpose of this study is to investigate the protective effect of alpha-linolenic acid (ALA) on Aβ25-35-induced neurotoxicity in C6 glial cells. Exposure of C6 glial cells to 50 μM Aβ25-35 caused cell death, overproduction of nitric oxide (NO), and pro-inflammatory cytokines release [interleukin (IL)-6 and tumor necrosis factor-α], while treatment of ALA increased cell viability and markedly attenuated Aβ25-35-induced excessive production of NO and those inflammatory cytokines. Inhibitory effect of ALA on generation of NO and cytokines was mediated by down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein and mRNA expressions. In addition, ALA treatment inhibited reactive oxygen species generation induced by Aβ25-35 through the enhancement of the nuclear factor-erythroid 2-related factor-2 (Nrf-2) protein levels and subsequent induction of heme-oxygenase-1 (HO-1) expression in C6 glial cells dose- and time-dependently. Furthermore, the levels of neprilysin and insulin-degrading enzyme protein expressions, which contribute to degradation of Aβ, were also increased by treatment of ALA compared to Aβ25-35-treated control group. In conclusion, effects of ALA on Aβ degradation were shown to be mediated through inhibition of inflammatory responses and activation of antioxidative system, Nrf-2/HO-1 signaling pathway, in C6 glial cells. Our findings suggest that ALA might have the potential for therapeutics of AD.

Modulation of the expression of genes related to the system of amyloid-beta metabolism in the brain as a novel mechanism of ceftriaxone neuroprotective properties

Background The dominant hypothesis about the pathogenesis of Alzheimer’s disease (AD) is the “amyloid cascade” concept and modulating the expression of proteins involved in the metabolism of amyloid-beta (Aβ) is proposed as an effective strategy for the prevention and therapy of AD. Recently, we found that an antibiotic ceftriaxone (CEF), which possesses neuroprotective activity, reduced cognitive deficits and neurodegenerative changes in OXYS rats, a model of sporadic AD. The molecular mechanisms of this effect are not completely clear, we suggested that the drug might serve as the regulator of the expression of the genes involved in the metabolism of Aβ and the pathogenesis of AD. The study was aimed to determine the effects of CEF on mRNA levels of Bace1 (encoding β-secretase BACE1 involved in Aβ production), Mme, Ide, Ece1, Ace2 (encoding enzymes involved in Aβ degradation), Epo (encoding erythropoietin related to endothelial function and clearance of Aβ across the blood brain barrier) in the frontal cortex, hippocampus, striatum, hypothalamus, and amygdala of OXYS and Wistar (control strain) male rats. Starting from the age of 14 weeks, animals received CEF (100 mg/kg/day, i.p., 36 days) or saline. mRNA levels were evaluated with RT-qPCR method. Biochemical parameters of plasma were measured for control of system effects of the treatment.ResultsTo better understand strain variations studied here, we compared the gene expression between untreated OXYS and Wistar rats. This comparison showed a significant decrease in mRNA levels of Ace2 in the frontal cortex and hypothalamus, and of Actb in the amygdala of untreated OXYS rats. Analysis of potential effects of CEF revealed its novel targets. In the compound-treated OXYS cohort, CEF diminished mRNA levels of Bace1 and Ace2 in the hypothalamus, and Aktb in the frontal cortex. Furthermore, CEF augmented Mme, Ide, and Epo mRNA levels in the amygdala as well as the levels of Ece1 and Aktb in the striatum. Finally, CEF also attenuated the activity of ALT and AST in plasma of OXYS rats.ConclusionThose findings disclosed novel targets for CEF action that might be involved into neuroprotective mechanisms at early, pre-plaque stages of AD-like pathology development.

Low‐Density Lipoprotein Receptor‐Related Protein 1 (LRP1) Is Degraded Primarily by The Lysosomal Degradation System in Hepatocellular Carcinoma (HepG2) Cells

LRP1 is a transmembrane protein responsible for binding and internalizing at least 50 different ligands, one of which is amyloid beta (Aβ). LRP1 has a role in clearance of brain and systemic Aβ. Understanding LRP1's role in Aβ degradation could have important implications for understanding Alzheimer's Disease (AD) pathogenesis. Since LRP1 in the liver is primarily responsible for systemic clearance of Aβ, its degradation could have important implications for AD pathogenesis.Normal degradation of proteins typically occurs through the ubiquitin proteasome system (UPS) and/or the lysosomal degradation system. To study LRP1 degradation through either of these two processes, one system was inhibited, then subsequent LRP1 output was analyzed. By inhibiting one degradation system at a time, we can analyze the protein output to quantify how much protein is degraded by each system. Bafilomycin A1 was used to inhibit the lysosomal degradation system, and MG132 was used to inhibit UPS. Finally, cycloheximide, a protein synthesis inhibitor, was used to determine LRP1 half‐life and to determine whether protein output is significantly affected by protein synthesis during inhibition of the protein degradation system (cycloheximide chase analysis).HepG2 cells were used to study LRP1 degradation in these experiments. The cells were treated with Bafilomycin A1, MG132, or dimethyl sulfoxide (DMSO; control group) and lysed at time points 0, 8, 16, and 24 hours to analyze LRP1 protein levels over time. Experiments with Bafilomycin A1 showed increasing levels of LRP1 over time, whereas experiments with MG132 showed unchanged, then decreasing levels of LRP1 over time. Cycloheximide was tested to determine the optimal concentration for HepG2 cells, which was found to be 30 μM. Then, experiments were performed with either cycloheximide and DMSO (as control), cycloheximide and Bafilomycin A1, or cycloheximide and MG132 at time points 0, 8, 16, and 24 hours to determine the half‐life of LRP1. Our results showed an extension of LRP1 half‐life by inhibition of lysosomal degradation system when using Bafilomycin A1, but half‐life was not extended when UPS was inhibited by MG132. Due to its significant increase in half‐life with Bafilomycin A1, but not with MG132, it's concluded that LRP1's degradation is primarily produced through the lysosomal degradation system.Support or Funding InformationMCPHS UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to β-amyloid (Aβ) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aβ binding. TREM2 deficiency impairs Aβ degradation in primary microglial culture and mouse brain. Aβ-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aβ, regulating downstream phosphorylation of SYK and GSK3β. Our data demonstrate TREM2 as a microglial Aβ receptor transducing physiological and AD-related pathological effects associated with Aβ.

Loss of kallikrein‐related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice

Deposition of amyloid‐β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimer's disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein‐related peptidase 7 (KLK7) as an astrocyte‐derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S‐positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatment in the primary astrocyte, suggesting that Klk7 is homeostatically modulated by Aβ‐induced responses. Finally, we found that the Food and Drug Administration‐approved anti‐dementia drug memantine can increase the expression of Klk7 and Aβ degradation activity specifically in the astrocytes. These data suggest that KLK7 is an important enzyme in the degradation and clearance of deposited Aβ species by astrocytes involved in the pathogenesis of AD.

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes.

Previous studies have shown that sirtuin 1 (SIRT1) reduces the production of neuronal amyloid beta (Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.

The effect of the neuroprotector isatin on complex formation of beta-amyloid peptide fragments with some intracellular proteins

Amyloid-β peptide (1-42) (Aβ1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aβ1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aβ1-42. It is reasonable to expect that dissociation of the Aβ1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aβ1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aβ1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aβ-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35). Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aβ1-42 and Aβ25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aβ peptides except Aβ1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aβ1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aβ12-28 and Aβ25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aβ studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aβ1-42, and its aggressive truncated peptides (Aβ25-35).

Nobiletin Reduces Intracellular and Extracellular β-Amyloid in iPS Cell-Derived Alzheimer's Disease Model Neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with progressive memory impairment. Recently, neprilysin, a β-amyloid (Aβ)-degrading enzyme has become featured as a drug target for AD. Previously, we identified nobiletin from citrus peels as a natural compound possessing anti-dementia activity. In addition, we demonstrated that nobiletin improved memory in memory-impaired animals and, further, that Aβ levels were markedly decreased in the brains of these animals. We demonstrated in vitro that nobiletin up-regulates neprilysin expression and activity in human neuroblastoma cells. However, the action of nobiletin with regard to Aβ degradation under in vitro AD pathological conditions remains unclear. In this study, we examined whether nobiletin could enhance the degradation of intra- and extracellular Aβ using human induced pluripotent stem cell-derived AD model neurons, which generate an excess of Aβ1-42 due to the familial AD presenilin-1 mutation. The neurons were treated in the presence or absence of nobiletin. The results of real-time quantitative RT-PCR indicated that neprilysin mRNA levels were significantly up-regulated by nobiletin. Furthermore, immunostaining with an anti-Aβ antibody revealed that nobiletin substantially reduced the intraneuronal content of Aβ. Interestingly, the results of Aβ1-42 immunoassays confirmed that nobiletin also significantly decreased the levels of Aβ1-42 released into the cellular medium. These results suggest that nobiletin enhanced the reduction of intra- and that extracellular Aβ levels under AD pathologic conditions, and this is associated with the up-regulation of neprilysin expression. Collectively, nobiletin appears to be a promising novel prophylactic seed drug or functional food for AD.

Reduced plaque size and inflammation in the APP23 mouse model for Alzheimer’s disease after chronic application of polymeric nanoparticles for CNS targeted zinc delivery

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer’s disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aβ), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aβ degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD.

Involvement of Astrocytes in Alzheimer’s Disease from a Neuroinflammatory and Oxidative Stress Perspective

Alzheimer disease (AD) is a frequent and devastating neurodegenerative disease in humans, but still no curative treatment has been developed. Although many explicative theories have been proposed, precise pathophysiological mechanisms are unknown. Due to the importance of astrocytes in brain homeostasis they have become interesting targets for the study of AD. Changes in astrocyte function have been observed in brains from individuals with AD, as well as in AD in vitro and in vivo animal models. The presence of amyloid beta (Aβ) has been shown to disrupt gliotransmission, neurotransmitter uptake, and alter calcium signaling in astrocytes. Furthermore, astrocytes express apolipoprotein E and are involved in the production, degradation and removal of Aβ. As well, changes in astrocytes that precede other pathological characteristics observed in AD, point to an early contribution of astroglia in this disease. Astrocytes participate in the inflammatory/immune responses of the central nervous system. The presence of Aβ activates different cell receptors and intracellular signaling pathways, mainly the advanced glycation end products receptor/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, responsible for the transcription of pro-inflammatory cytokines and chemokines in astrocytes. The release of these pro-inflammatory agents may induce cellular damage or even stimulate the production of Aβ in astrocytes. Additionally, Aβ induces the appearance of oxidative stress (OS) and production of reactive oxygen species and reactive nitrogen species in astrocytes, affecting among others, intracellular calcium levels, NADPH oxidase (NOX), NF-κB signaling, glutamate uptake (increasing the risk of excitotoxicity) and mitochondrial function. Excessive neuroinflammation and OS are observed in AD, and astrocytes seem to be involved in both. The Aβ/NF-κB interaction in astrocytes may play a central role in these inflammatory and OS changes present in AD. In this paper, we also discuss therapeutic measures highlighting the importance of astrocytes in AD pathology. Several new therapeutic approaches involving phenols (curcumin), phytoestrogens (genistein), neuroesteroids and other natural phytochemicals have been explored in astrocytes, obtaining some promising results regarding cognitive improvements and attenuation of neuroinflammation. Novel strategies comprising astrocytes and aimed to reduce OS in AD have also been proposed. These include estrogen receptor agonists (pelargonidin), Bambusae concretio Salicea, Monascin, and various antioxidatives such as resveratrol, tocotrienol, anthocyanins, and epicatechin, showing beneficial effects in AD models.

A mechanistic model to predict effects of cathepsin B and cystatin C on β-amyloid aggregation and degradation

β-Amyloid (Aβ) aggregation is thought to initiate a cascade of neurodegenerative events in Alzheimer's disease (AD). Much effort is underway to develop strategies to reduce Aβ concentration or inhibit aggregation. Cathepsin B (CatB) proteolytically degrades Aβ into non-aggregating fragments but is potently inhibited by cystatin C (CysC). It has been suggested that decreasing CysC would facilitate Aβ clearance by relieving CatB inhibition. However, CysC binds Aβ and inhibits Aβ aggregation, suggesting that an intervention that increases CysC would prevent Aβ aggregation. Both approaches have been tested in animal models, yielding contradictory results, possibly because of the opposing influences of CysC on Aβ degradation versus aggregation. Here, we sought to develop a model that quantitatively predicts the effects of CysC and CatB on Aβ aggregation. Aβ aggregation kinetics in the absence of CatB or CysC was measured. The rate constant for Aβ degradation by CatB and the equilibrium constant for binding of CysC to Aβ were determined. We derived a mathematical model that combines material balances and kinetic rate equations. The model accurately predicted Aβ aggregation kinetics at various CatB and CysC concentrations. We derived approximate expressions for the half-times of degradation and aggregation and show that their ratio can be used to estimate, at any given Aβ, CatB, or CysC concentration, whether Aβ aggregation or degradation will result. Our results may be useful for designing experiments and interpreting results from investigations of manipulation of CysC concentration as an AD therapy.

Alpha-Linolenic Acid from Perilla frutescens var. japonica Oil Protects Aβ-Induced Cognitive Impairment through Regulation of APP Processing and Aβ Degradation.

Alzheimer's disease (AD) is characterized by progressive cognitive and memory impairment. The major pathological hallmark of AD is the accumulation of amyloid beta (Aβ), which is produced from the amyloid precursor protein (APP) through cleavage of β- and γ-secretase. Recently, dietary plant oil containing ω-3 polyunsaturated fatty acid has become an attractive alternative source to fish oil containing eicosapentaenoic acid or docosahexaenoic acid (DHA). We investigated whether ALA isolated from perilla oil has direct effects on improvement of cognitive ability and molecular mechanisms in APP processing in comparison with DHA. In the present study, ICR mice were treated orally with ALA or DHA (100 mg/kg/day) for 14 days after i.c.v. injection of Aβ25-35. Administration of ALA resulted in a prevention of learning and memory deficit in Aβ25-35-injected mice compared with the control group, as observed in T-maze, novel object recognition, and Morris water maze tests. ALA supplementation also markedly ameliorated the Aβ25-35-induced oxidative stress by inhibition of lipid peroxidation and nitric oxide overproduction in the mouse brain, liver, and kidney, almost down to the levels in DHA-administered group. These effects of ALA on protective mechanisms were related to the regulation of APP processing via promoting nonamyloidogenic pathway such as up-regulation of soluble APP alpha, C-terminal fragment alpha/beta ratio, and A disintegrin and metalloprotease10 protein expressions. Furthermore, ALA inhibited the amyloidogenic pathway through the down-regulation of β-site APP-cleaving enzyme and presenilin2. ALA also enhanced Aβ degradation enzyme, insulin-degrading enzyme. In conclusion, the present study indicated a beneficial effect of ALA in improving the cognitive ability against Aβ25-35, and these effects were comparable to those exerted by DHA. Its neuroprotective effects are mediated, in part, by regulation of APP processing and Aβ degradation, and thus, ALA might be a potential candidate for prevention or treatment of neurodegenerative diseases such as AD.

Androgen alleviates neurotoxicity of β-amyloid peptide (Aβ) by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ.

Lower androgen level in elderly men is a risk factor of Alzheimer's disease (AD). It has been reported that androgen reduces amyloid peptides (Aβ) production and increases Aβ degradation by neurons. Activated microglia are involved in AD by either clearing Aβ deposits through uptake of Aβ or releasing cytotoxic substances and pro-inflammatory cytokines. Here, we investigated the effect of androgen on Aβ uptake and clearance and Aβ-induced inflammatory response in microglia, on neuronal death induced by Aβ-activated microglia, and explored underlying mechanisms. Intracellular and extracellular Aβ were examined by immunofluorescence staining and Western blot. Amyloid peptides (Aβ) receptors, Aβ degrading enzymes, and pro-inflammatory cytokines were detected by RT-PCR, real-time PCR, and ELISA. Phosphorylation of MAP kinases and NF-κB was examined by Western blot. We found that physiological concentrations of androgen enhanced Aβ42 uptake and clearance, suppressed Aβ42 -induced IL-1β and TNFα expression by murine microglia cell line N9 and primary microglia, and alleviated neuronal death induced by Aβ42 -activated microglia. Androgen administration also reduced Aβ42 -induced IL-1β expression and neuronal death in murine hippocampus. Mechanistic studies revealed that androgen promoted microglia to phagocytose and degrade Aβ42 through upregulating formyl peptide receptor 2 and endothelin-converting enzyme 1c expression, and inhibited Aβ42 -induced pro-inflammatory cytokines expression via suppressing MAPK p38 and NF-κB activation by Aβ42 , in an androgen receptor independent manner. Our study demonstrates that androgen promotes microglia to phagocytose and clear Aβ42 and inhibits Aβ42 -induced inflammatory response, which may play an important role in reducing the neurotoxicity of Aβ.

Thymoquinone-rich fraction nanoemulsion (TQRFNE) decreases Aβ40 and Aβ42 levels by modulating APP processing, up-regulating IDE and LRP1, and down-regulating BACE1 and RAGE in response to high fat/cholesterol diet-induced rats

Though the causes of Alzheimer’s disease (AD) are yet to be understood, much evidence has suggested that excessive amyloid-β (Aβ) accumulation due to abnormal amyloid-β precursor protein (APP) processing and Aβ metabolism are crucial processes towards AD pathogenesis. Hence, approaches aiming at APP processing and Aβ metabolism are currently being actively pursued for the management of AD. Studies suggest that high cholesterol and a high fat diet have harmful effects on cognitive function and may instigate the commencement of AD pathogenesis. Despite the neuropharmacological attributes of black cumin seed (Nigella sativa) extracts and its main active compound, thymoquinone (TQ), limited records are available in relation to AD research. Nanoemulsion (NE) is exploited as drug delivery systems due to their capacity of solubilising non-polar active compounds and is widely examined for brain targeting. Herewith, the effects of thymoquinone-rich fraction nanoemulsion (TQRFNE), thymoquinone nanoemulsion (TQNE) and their counterparts’ conventional emulsion in response to high fat/cholesterol diet (HFCD)-induced rats were investigated. Particularly, the Aβ generation; APP processing, β-secretase 1 (BACE1), γ-secretases of presenilin 1 (PSEN1) and presenilin 2 (PSEN2), Aβ degradation; insulin degrading enzyme (IDE), Aβ transportation; low density lipoprotein receptor-related protein 1 (LRP1) and receptor for advanced glycation end products (RAGE) were measured in brain tissues. TQRFNE reduced the brain Aβ fragment length 1–40 and 1–42 (Aβ40 and Aβ42) levels, which would attenuate the AD pathogenesis. This reduction could be due to the modulation of β- and γ-secretase enzyme activity, and the Aβ degradation and transportation in/out of the brain. The findings show the mechanistic actions of TQRFNE in response to high fat and high cholesterol diet associated to Aβ generation, degradation and transportation in the rat’s brain tissue.

Epigallocatechin gallate induces extracellular degradation of amyloid β-protein by increasing neprilysin secretion from astrocytes through activation of ERK and PI3K pathways

Amyloid-β (Aβ) production and clearance in the brain is a crucial focus of investigations into the pathogenesis of Alzheimer disease. Imbalance between production and clearance leads to accumulation of Aβ. The important Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), and defective enzyme expression may facilitate Aβ deposition in sporadic late-onset AD patients. It has been suggested that epigallocatechin gallate (EGCG), a member of the catechin family, might be an effective treatment for AD, because it has been shown to elevate NEP expression. Therefore, we examined whether catechins, which are functional components of common foods, could regulate the degradation of Aβ by inducing NEP and IDE expression. We also investigated the role of catechins in activating intracellular signal transduction in astrocytes. Treatment of cultured rat astrocytes with EGCG significantly reduced the expression of NEP, but not IDE, in a concentration- and time-dependent manner. NEP expression in cultured astrocytes was suppressed by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), and reduced NEP expression was accompanied by an increase of NEP release into the extracellular space (culture medium). Moreover, culture medium from EGCG-treated astrocytes facilitated the degradation of exogenous Aβ. These results suggest that EGCG may have a beneficial effect on AD by activating ERK-and PI3K-mediated pathways in astrocytes, thus increasing astrocyte secretion of NEP and facilitating degradation of Aβ.

Differential protective effects of connective tissue growth factor against Aβ neurotoxicity on neurons and glia.

Impaired clearance of amyloid-β peptide (Aβ) leads to abnormal extracellular accumulation of this neurotoxic protein that drives neurodegeneration in sporadic Alzheimer's disease (AD). Connective tissue growth factor (CTGF/CCN2) expression is elevated in plaque-surrounding astrocytes in AD patients. However, the role of CTGF in AD pathogenesis remains unclear. Here we characterized the neuroprotective activity of CTGF. We found that CTGF facilitated Aβ uptake and subsequent degradation within primary glia and neuroblastoma cells. CTGF enhanced extracellular Aβ degradation via membrane-bound matrix metalloproteinase-14 (MMP14) in glia and extracellular MMP13 in neurons. In the brain of a Drosophila AD model, glial-expression of CTGF reduced Aβ deposits, improved locomotor function, and rescued memory deficits. Neuroprotective potential of CTGF against Aβ42-induced photoreceptor degeneration was disrupted through silencing MMPs. Therefore, CTGF may represent a node for potential AD therapeutics as it intervenes in glia-neuron communication via specific MMPs to alleviate Aβ neurotoxicity in the central nervous system.

REVERSAL OF LYSOSOMAL DYSFUNCTION RESCUES BETA-AMYLOID PATHOGENESIS IN ALZHEIMER’S DISEASE

Amyloid beta (Aβ) deposit is a major neuropathological hallmark of Alzheimer's disease (AD). It has been identified that intracellular Aβ (iAβ) accumulation is one of the crucial events in AD pathogenesis. Amount of evidence suggest that insufficient cellular clearance of iAβ results from lysosomal dysfunctions and enhancement of lysosomal function restores AD pathology. Therefore, identifying genes that promote the clearance of iAβ by reinforcing lysosomal activity and discovering the underlying mechanism are vital for AD intervention. For cell-based functional screening, cells were transfected with cDNA encoding lysosome-associated proteins and then further incubated with synthetic oligomeric Aβ1–42. Cytotoxicity was measured by staining with EthD. Hippocampal homogenates from AD patients with Braak stage V or VI and non-AD patients were provided by the Harvard Brain Tissue Resource Center (McLean Hospital, Belmont, Massachusetts, USA). For detection of intracellular Aβ1–42, membrane-associated Aβ1–42were washed with trypsin-EDTA and harvested cells were subjected to immunoblotting using 4G8 and 6E10 antibodies. Activity-defective mutant HYAL D129A/E131A was generated by mutagenesis. For lysosomal activity assay using fusion protein degraded by lysosome, GFP-CINCCKVL was amplified from pEGFP-C1 vector using synthesized primers (EGFP-5′-HindIII, EGFP-CINCCKVL-3′-EcoRI) and subcloned into pcDNA3.0. HYAL was isolated as the most effective gene that suppressed Aβ-induced cell death. Compared to normal controls, HYAL expression was significantly decreased in AD brains. Downregulation of HYAL aggravated Aβ-induced cell death and increased iAβ level. In overexpression assay, HYAL dramatically decreased Aβ-neurotoxicity and reduced iAβ level. An activity-defective mutant altered neither Aβ-induced toxicity nor the iAβ level, indicating that catalytic activity is critical for its protective role. Degradation of iAβ was halted by lysosomal inhibitors but not by proteasomal inhibitor, indicating that iAβ is degraded mostly by lysosomal pathway. In lysosomal activity assay, ectopically expressed HYAL hindered abnormal accumulation of lysosomal substrates due to Aβ1–42 exposure, suggesting that HYAL serve as positive lysosomal regulators in Aβ pathogenesis. These results suggest that HYAL exerts a protective role by facilitating Aβ degradation, possibly through enhancing lysosomal function. Reference: (1) Frank M. et al. Nat. Rev. Neurosci. 2007; 430(7000):499–509. (2) Nixon, R.A., and Yang, D.-S. Neurobiol. Dis. 2011; 43, 38–45.