Oleracone F Alleviates Cognitive Impairment and Neuropathology in APPswe/PSEN1dE9 Mice by Reducing the Expression of Vascular Cell Adhesion Molecule and Leukocyte Adhesion to Brain Vascular Endothelial Cells.

Alzheimer's disease (AD) is the most common neurodegenerative disease and the blood-brain barrier dysfunction has been suggested as a key pathological feature of the disease. Our research group successfully established a synthetic protocol for oleracones, a novel series of flavonoids isolated from the plant extract of Portulaca oleracea L. (PO). PO extract was reported to have anti-inflammatory and antioxidant effects, enhancing cognitive function. Thus, we investigated the effects and mechanism of oleracones on cognition using AD model transgenic mice (Tg; APPswe/PSEN1dE9). Oleracone F treatment significantly improved memory dysfunction in Tg mice. Oleracone F decreased the number, burden, and immunoreactivity of amyloid plaques and amyloid precursor protein (APP) protein levels in the brains of Tg mice compared to wild-type mice. Oleracone F also alleviated inflammation observed in Tg mice brains. In vitro studies in human microvascular endothelial cells (HBMVECs) demonstrated that oleracones D, E, and F blocked the elevations in VCAM-1 protein induced by tumor necrosis factor-α (TNF-α), hindering leukocyte adhesion to HBMVECs. Taken together, our results suggest that oleracones ameliorated cognitive impairment by blocking TNF-α-induced increases in VCAM-1, thereby reducing leukocyte infiltration to the brain and modulating brain inflammation.

HDAC1 regulates the stability of glutamate carboxypeptidase II protein by modulating acetylation status of lysine 479 residue.

Our previous study showed that the level of glutamate carboxypeptidase II (GCPII) protein is regulated by valproic acid, a histone deacetylase (HDAC) inhibitor, through acetylation of lysine residue in the GCPII protein in human astrocytes, U-87MG. The present study further investigated which HDAC subtype is involved in the acetylation of GCPII. The results revealed that GCPII interacted with HDAC1 but not with HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6. Overexpression of catalytic domain (1-56 aa)-deleted HDAC1, which poorly binds to GCPII, enhanced lysine acetylation in GCPII and increased the level of GCPII protein when compared with that of the wild-type HDAC1. Further experiments showed that HDAC1 regulated the stability of GCPII protein. These data suggest that acetylation of GCPII is facilitated by HDAC1, and the acetylated GCPII is more stable than the non-acetylated GCPII. Additional experiments using siRNA HDAC1 and by HDAC1 overexpression confirmed the role of HDAC1 in regulating the stability of GCPII protein. Further, database search of acetylation and ubiquitination sites showed four candidate lysine sites in human GCPII protein that can be both acetylated and ubiquitinylated (K207, K479, K491, and K699). Mutation (lysine residues to arginine (R)) analysis showed that in the presence of cycloheximide K479R- and K491R-hGCPII mutants were less ubiquitinylated and degraded, and decrease in the level of GCPII protein by HDAC1 was significantly blocked by K479R mutants. These data suggest that K479 is a possible site of acetylation or ubiquitination. Furthermore, the results also demonstrate that the stability of GCPII protein is regulated by HDAC1 through acetylation at the lysine 479 residue.

Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass.

BACKGROUND AND AIMS: Osteoporosis, which is a disease characterized by weakening of the bone, affects a large portion of the senior population. The current therapeutic options for osteoporosis have side effects, and there is no effective treatment for severe osteoporosis. Thus, we urgently need new treatment strategies, such as topical therapies and/or safe and effective stem cell therapies. METHODS: We investigated the therapeutic potential of directly injecting human tonsil-derived mesenchymal stem cells (TMSC) into the right proximal tibias of ovariectomized postmenopausal osteoporosis model mice. Injections were given once (1×) or twice (2×) during the 3-month experimental period. At the end of the experiment, micro-computed tomographic images revealed some improvement in the proximal tibias and more significant improvement in the femoral heads of treated mice. RESULTS: Osteogenic effect was qualitatively and quantitatively more pronounced in TMSC/2×-treated mice. Furthermore, TMSC/2× mice exhibited significant recovery of the serum osteocalcin level, which is pathologically elevated in osteoporosis, and increased serum alkaline phosphatase, which indicates bone formation. TMSC therapy was generally well tolerated and caused no apparent toxicity in the experimental mice. Moreover, TMSC therapy reduced visceral fat. CONCLUSION: Our results demonstrate that double injection of TMSC directly into the proximal tibia triggers recovery of osteoporosis, and thus could be a potential therapeutic approach for severe bone loss.

KDM7A histone demethylase mediates TNF-α-induced ICAM1 protein upregulation by modulating lysosomal activity.

Intercellular adhesion molecule1 (ICAM1) is involved in adhesion and transmigration of leukocytes across endothelium, promoting brain inflammation and leading to brain diseases. Here, we studied the mechanism that regulates ICAM expression in response to proinflammatory cytokine tumor necrosis factor alpha (TNF-α). ICAM1 mRNA and protein levels in human brain microvascular endothelial cells dramatically increased after TNF-α treatment. TNF-α also upregulated histone demethylases KDM1B and KDM7A responsible for demethylation of H3K9me2, a well-known repression marker. Knockdown of KDM7A by a small interfering RNA reduced the ICAM1 protein level and leukocyte adhesion without an effect on ICAM1 mRNA expression. In contrast, a KDM1B knockdown did not affect TNF-α-induced ICAM1 expression. Thus, KDM7A-mediated ICAM1 protein upregulation is likely related to protein stability, not a histone-mediated epigenetic mechanism. Experiments with cycloheximide supported the role of KDM7A in ICAM1 protein stabilization. Further experiments suggest that KDM7A regulates ICAM1 protein stability via a lysosome-dependent pathway. Lysosome inhibitors increased the TNF-α-induced ICAM1 protein level and restored KDM7A knockdown-induced downregulation of ICAM1. In contrast, the KDM7A knockdown had no effect on proteasome-mediated ICAM1 degradation. We also found that the transcription factor EB protein level reduced in response to TNF-α but increased by the KDM7A knockdown. Immunocytochemical analysis revealed weak lysosome formation with high ICAM1 immunoreactivity after TNF-α treatment, but KDM7A knockdown reversed this response, resulting in strong lysosome formation with ICAM1 protein clustering in lysosomes. Taken together, our results show that KDM7A mediates TNF-α-induced ICAM1 protein upregulation and is mediated by induction of KDM7A, which regulates the TFEB-mediated lysosomal activity.

Acetylation regulates the stability of glutamate carboxypeptidase II protein in human astrocytes.

Glutamate carboxypeptidase II (GCPII) is known to be implicated in brain diseases such as schizophrenia and bipolar disorder, and dramatically increases in prostate cancer. Here, we investigated the regulation of GCPII expression in astrocytes and examined whether GCPII is epigenetically regulated through histone modification. In this study, valproic acid (VPA), a drug used for bipolar disorder and epilepsy and a known histone deacetylase (HDAC) inhibitor was used. We found that acute exposure of VPA for 4-6h increased the GCPII protein level in human astrocyte U87MG cells but did not have a similar effect after 12-24h exposure. Real-time polymerase chain reaction analysis revealed that VPA did not affect the GCPII mRNA expression. In contrast, decrease in GCPII protein level by cycloheximide treatment was blocked by VPA, indicating that VPA increases GCPII protein stability. Treatment with MG132, a proteasome inhibitor, suggested that the VPA-induced increase of GCPII protein level is dependent on the ubiquitin/proteasome pathway. In addition, immunoprecipitation analysis revealed that VPA increased the acetylation of GCPII protein at the lysine residues and facilitated a decrease of the poly-ubiquitinated GCPII level. Similarly, M344, a specific HDAC 1/6 inhibitor, also increased the GCPII protein level. In contrast, treatment with C646, a histone acetyltransferase inhibitor of p300/CBP, significantly reduced the level of GCPII protein. Taken together, this study demonstrated that the increase in GCPII induced by VPA is not due to the classical epigenetic mechanism, but via enhanced acetylation of lysine residues in GCPII.

Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.

5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.

Exercise ameliorates cognition impairment due to restraint stress-induced oxidative insult and reduced BDNF level.

We assessed whether chronic treadmill exercise attenuated restraint stress-induced cognition impairment. Although serum corticosterone was not significantly altered by exercise, the restraint-induced increases in hippocampal malondialdehyde (MDA) and 4-hydroxynonenal (HNE) were reduced by chronic exercise. The exercise paradigm also reversed stress-induced reductions in brain-derived neurotrophic factor (BDNF), which increased cAMP response element-binding protein (CREB) and AKT activation. We verified the relationship between oxidative stress and BDNF signaling by treating primary hippocampal cultures with hydrogen peroxide (H2O2), which reduced BDNF and phosphorylated CREB and AKT (p-CREB, p-AKT) in a dose-dependent manner. Notably, pretreatment with N-acetylcysteine (NAC) reversed these decreases in a dose-dependent manner. These findings suggest that chronic exercise can ameliorate repeated stress-induced cognitive impairment by detoxifying reactive oxygen species (ROS) in the hippocampus and activating BDNF signaling.

S1 pocket of glutamate carboxypeptidase II: a new binding site for amyloid-ß degradation.

We recently reported that glutamate carboxypeptidase II (GCPII) has a new physiological function degrading amyloid-ß (Aß), distinct from its own hydrolysis activity in N-acetyl-L-aspartyl-L-glutamate (NAAG); however, its underlying mechanism remains undiscovered. Using site-directed mutagenesis and S1 pocket-specific chemical inhibitor (compound 2), which was developed for the present study based on in sillico computational modeling, we discovered that the Aß degradation occurs through S1 pocket but not through S1' pocket responsible for NAAG hydrolysis. Treatment with compound 2 prevented GCPII from Aß degradation without any impairment in NAAG hydrolysis. Likewise, 2-PMPA (specific GCPII inhibitor developed targeting S1' pocket) completely blocked the NAAG hydrolysis without any effect on Aß degradation. Pre-incubation with NAAG and Aß did not affect Aß degradation and NAAG hydrolysis, respectively. These data suggest that GCPII has two distinctive binding sites for two different substrates and that Aß degradation occurs through binding to S1 pocket of GCPII.

Increased carotid intima-media thickness and plasma homocysteine levels predict cardiovascular and all-cause death: a population-based cohort study.

BACKGROUND: There is still debate over the utility of carotid intima-media thickness (C-IMT) or carotid plaque in predicting future cardiovascular events and death. Additionally, the importance of plasma homocysteine levels was raised as a predictor of cardiovascular events and death. METHODS: 1,391 subjects were recruited from the Ansan Geriatric cohort. We used B-mode carotid ultrasonography to assess C-IMT and plaque, measuring average maximal IMT and average mean IMT through 6-8 measurements of far-wall IMT in both common carotid arteries. We evaluated the presence of plaque in carotid segments. Multivariable Cox regression analysis was used to predict both cardiovascular and all-cause mortality. RESULTS: During a mean follow-up of 62.4 ± 12.4 months, 71 subjects (5.12%) died and 23 (1.66%) died of cardiovascular causes. Multivariable Cox regression analysis found the predictors of cardiovascular mortality to be average maximal IMT (HR = 3.709; 95% CI: 1.202-11.446) and plasma homocysteine (HR = 1.057; 95% CI: 1.012-1.103). All-cause mortality was independently associated with C-IMT (average maximal and average mean IMT) and plasma homocysteine. CONCLUSIONS: C-IMT and plasma homocysteine levels were found to predict cardiovascular and all-cause mortality independently of the presence of carotid plaque and other cardiovascular risk factors.

Induction of ICAM1 in Brain Vessels is Implicated in an Early AD Pathogenesis by Modulating Neprilysin.

Intercellular adhesion molecule 1 (ICAM1) is a vessel adhesion protein induced during brain vascular inflammation, which could be closely linked with the development of Alzheimer's disease (AD). This study investigated the effect of ICAM1 on amyloid-degrading enzymes (ADEs) in endothelial cells and their potential involvement in inflammation and AD progression. TNF-α treatment increased ICAM1 in human brain microvascular endothelial cells (HBMVECs) but decreased the neprilysin (NEP) protein level. Knock-down of ICAM1 using siRNA enhanced NEP, which increased the degradation of amyloid-ß. In the brains of 4-month-old AD transgenic mice (APPswe/PSEN1dE9), there were significantly higher levels of ICAM1 expression and amyloid deposits but lower levels of NEP and insulin-degrading enzymes (IDE), demonstrating an inverse correlation of ICAM1 with NEP and IDE expression. Further studies demonstrated significantly increased GFAP protein levels in the brain, specifically localized near blood vessels, of both TNF-α-injected and 4-month-old AD transgenic mice. Taken together, the induction of ICAM1 in endothelial cells suppresses NEP expression, accelerating the accumulation of amyloid-ß in blood vessels. It also enhances leukocyte adhesion to blood vessels stimulating the migration of leukocytes into the brain, subsequently triggering brain inflammation.

Mitogen-activated protein kinase kinase 1 (MEK1) stabilizes MyoD through direct phosphorylation at tyrosine 156 during myogenic differentiation.

Previously, we reported that mitogen-activated protein kinase kinase 1 (MEK1) activated in the mid-stage of skeletal muscle differentiation promotes myogenic differentiation. To elucidate the molecular mechanism, we investigated an activity of MEK1 for MyoD. Activated MEK1 associates with MyoD in the nucleus of differentiating myoblasts. In vitro kinase assay using active MEK1, a (32)P-labeled protein band corresponding to GST-MyoD was observed but not to mutant GST-MyoD-Y156F. Tyrosine phosphorylation of endogenous MyoD was detected with a specific anti-pMyoD-Y156 antibody; however, this response was blocked by PD184352, a MEK-specific inhibitor. These results indicate that activated MEK1 phosphorylates the MyoD-Y156 residue directly. Interestingly, the protein level of mutant MyoD-Y156F decreased compared with that of wild type but was recovered in the presence of lactacystin, a proteasome inhibitor. The protein level of MyoD-Y156E, which mimics phosphorylation at Tyr-156, was above that of wild type, indicating that the phosphorylation protects MyoD from the ubiquitin proteasome-mediated degradation. In addition, the low protein level of MyoD-Y156F was recovered over that of wild type by an additional mutation at Leu-164, a critical binding residue of MAFbx/AT-1, a Skp, Cullin, F-box (SCF) E3-ubiquitin ligase. The amount of MyoD co-precipitated with MAFbx/AT-1 also was reduced in the presence of active MEK1. Thus, these results suggested that the phosphorylation probably interrupts the binding of MAFbx/AT-1 to MyoD and thereby increases its stability. Collectively, our results suggest that MEK1 activated in differentiating myoblasts stimulates muscle differentiation by phosphorylating MyoD-Y156, which results in MyoD stabilization.

DNA methylation of the 5'-untranslated region at +298 and +351 represses BACE1 expression in mouse BV-2 microglial cells.

BACE1, which cleaves the amyloid precursor protein, is the rate-limiting enzyme for ß-amyloid peptide production, leading to the pathogenesis of Alzheimer's disease (AD). A high plasma level of homocysteine, acting as a potent methyltransferase inhibitor, is assumed to be a risk factor for AD onset. Using the demethylating drug 5-aza-2'-deoxycytidine (5-Aza), we tested whether and how BACE1 expression is regulated in mouse BV-2 microglial cells. 5-Aza increased both BACE1 mRNA and protein levels in a dose-dependent manner. Bisulfite-sequencing analysis revealed that two CpG sites at positions +298 and +351 in the 5'-untranslated region (5'-UTR) of the BACE1 gene were specifically demethylated in BV-2 cells treated with 5-Aza. In silico analysis showed that the +351 site is the STAT3/CTCF-binding site; the function of the +298 site has not been identified. To assess whether these two CpG sites play an important role in 5-Aza-induced transcriptional activation of BACE1, we constructed a BACE1 gene promoter including the 5'-UTR (-1136 to +500) fused to a CpG-free luciferase gene (pCpGL-BACE1) and its mutant pCpGL-BACE1-AA, which has substituted CG dinucleotides at the two CpG sites of pCpGL-BACE1 to AA. Promoter analysis showed a significant decrease (∼30%) in the activity of pCpGL-BACE1-AA compared with that of pCpGL-BACE1. Furthermore, in vitro methylation of these two reporter constructs showed a complete silencing of their promoter activities. Our data demonstrate that BACE1 gene expression is regulated by DNA methylation of at least two CpG sites at positions +298 and +351 in the 5'-UTR in BV-2 microglial cells.

Choline acetyltransferase 2384G>a polymorphism and the risk of Alzheimer disease.

The potential association between choline acetyltransferase (CHAT) polymorphism and the risk of Alzheimer disease (AD) has been controversial. We examined the main effect of CHAT polymorphism and its interaction with apolipoprotein E (APOE) polymorphism in the development of AD in a well-powered elderly Korean sample. We analyzed CHAT 2384G>A polymorphism and APOE polymorphism among 736 Korean patients with probable AD and 1386 nondemented Korean controls. We tested the association between AD and CHAT genotype using a logistic regression model. In addition, we used generalized multifactor dimensionality reduction to investigate the interaction between CHAT and APOE with regard to the risk of AD. The CHAT A allele was associated with AD risk in a dose-dependent manner (odds ratio=1.40, 95% confidence interval=1.06-1.85, P=0.018 for heterozygotes; and odds ratio=3.92, 95% confidence interval=1.78-8.58, P=0.001 for homozygotes). The generalized multifactor dimensionality reduction approach identified a significant gene-gene interaction between CHAT and APOE (Balanced accuracy score=0.647, P=0.001). The CHAT A/A genotype was associated with earlier onset of AD (F=5.070, df=2, P=0.007). The CHAT A allele was associated with AD risk in a dose-dependent manner, and its interaction with the APOE ε4 allele was significant with regard to the development of AD. The CHAT A allele was also associated with earlier onset and possibly accelerated progression of AD.

Basic Fibroblast Growth Factor Induces Cholinergic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are considered a potential tool for regenerating damaged tissues due to their great multipotency into various cell types. Here, we attempted to find the appropriate conditions for neuronal differentiation of tonsil-derived MSCs (TMSCs) and expand the potential application of TMSCs for treating neurological diseases. METHODS: The TMSCs were differentiated in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) supplemented with various neurotrophic factors for 7-28 days to determine the optimal neuronal differentiation condition for the TMSCs. The morphologies as well as the levels of the neural markers and neurotransmitters were assessed to determine neuronal differentiation potentials and the neuronal lineages of the differentiated TMSCs. RESULTS: Our initial study demonstrated that DMEM/F12 supplemented with 50 ng/mL basic fibroblast growth factor with 10 µM forskolin was the optimal condition for neuronal differentiation for the TMSCs. TMSCs had higher protein expression of neuronal markers, including neuron-specific enolase (NSE), GAP43, postsynaptic density protein 95 (PSD95), and synaptosomal-associated protein of 25 kDa (SNAP25) compared to the undifferentiated TMSCs. Immunofluorescence staining also validated the increased mature neuron markers, NeuN and synaptophysin, in the differentiated TMSCs. The expression of glial fibrillar acidic protein and ionized calcium-binding adaptor molecule 1 the markers of astrocytes and microglia, were also slightly increased. Additionally, the differentiated TMSCs released a significantly higher level of acetylcholine, the cholinergic neurotransmitter, as analyzed by the liquid chromatography-tandem mass spectrometry and showed an enhanced choline acetyltransferase immunoreactivity compared to the undifferentiated cells. CONCLUSION: Our study suggests that the optimized condition favors the TMSCs to differentiate into cholinergic neuron-like phenotype, which could be used as a possible therapeutic tool in treating certain neurological disorders such as Alzheimer's disease.

Effects of Natural Polyphenols on Oxidative Stress-Mediated Blood-Brain Barrier Dysfunction.

The blood-brain barrier (BBB), which consists mainly of brain microvascular endothelial cells and astrocytes connected by tight junctions (TJs) and adhesion molecules (AMs), maintains the homeostatic balance between brain parenchyma and extracellular fluid. Accumulating evidence shows that BBB dysfunction is a common feature of neurodegenerative diseases, including stroke, traumatic brain injury, and Alzheimer's disease. Among the various pathological pathways of BBB dysfunction, reactive oxygen species (ROS) are known to play a key role in inducing BBB disruption mediated via TJ modification, AM induction, cytoskeletal reorganization, and matrix metalloproteinase activation. Thus, antioxidants have been suggested to exert beneficial effects on BBB dysfunction-associated brain diseases. In this review, we summarized the sources of ROS production in multiple cells that constitute or surround the BBB, such as BBB endothelial cells, astrocytes, microglia, and neutrophils. We also reviewed various pathological mechanisms by which BBB disruption is caused by ROS in these cells. Finally, we summarized the effects of various natural polyphenols on BBB dysfunction to suggest a therapeutic strategy for BBB disruption-related brain diseases.

Valproic Acid-Induced CCN1 Promotes Osteogenic Differentiation by Increasing CCN1 Protein Stability through HDAC1 Inhibition in Tonsil-Derived Mesenchymal Stem Cells.

Our previous study found that the level of CCN1 increases as osteogenic differentiation progresses in tonsil-derived mesenchymal stem cells (TMSCs). This study investigated how CCN1 is regulated through HDAC inhibition in TMSCs and their relationship with osteogenesis. Valproic acid (VPA) (1-5 mM), a well-known histone deacetylase (HDAC) inhibitor, strongly inhibited TMSC proliferation without altering MSC-specific surface markers, CD14, 34, 45, 73, 90 and 105. However, CD146 expression increased at 5 mM VPA. VPA increased osteogenic differentiation of TMSCs but decreased adipogenesis and chondrogenesis, as evidenced by the cell-specific staining of differentiation. The former was validated by the increased osteocalcin (OCN). The changes in CCN1 by VPA was biphasic; it increased until 48 h and decreased thereafter. Knockdown of CCN1 by using siRNA inhibited the osteogenic effect of VPA. VPA had no effect on CCN1 mRNA expression, but inhibition of protein synthesis by cycloheximide showed that VPA slowed down the CCN1 protein degradation. Moreover, overexpression of HDAC1 completely inhibited VPA-induced CCN1. Our results indicate that VPA inhibits the HDAC1, inducing CCN1 protein stability rather than gene expression, thereby promoting osteogenic differentiation of TMSCs. These findings present the noble implication of VPA as an inhibitor of HDAC1 to facilitate CCN1-induced osteogenic differentiation of MSCs.

2,4-Diacetylphloroglucinol Reduces Beta-Amyloid Production and Secretion by Regulating ADAM10 and Intracellular Trafficking in Cellular and Animal Models of Alzheimer's Disease.

There is currently no effective treatment against Alzheimer's disease (AD), although many strategies have been applied to reduce beta-amyloid (Aß) levels. Here, we investigated 2,4-diacetylphloroglucinol (DAPG) effects on Aß levels and mechanisms of action. DAPG was the most effective phloroglucinol derivative for reducing Aß levels, without being toxic, in various models including HEK293 cells overexpressing Swedish mutant amyloid precursor protein (APP) (293sw), primary astrocytes isolated from APPsw/PS1dE9 transgenic mice, and after intrahippocampal injection of DAPG in APPsw/PS1dE9 transgenic mice. DAPG-mediated Aß reduction was associated with increased soluble APPα (sAPPα) levels mediated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) but not ADAM17. ADAM10 inhibition in DAPG-treated cells prevented the effects on sAPPα but only partly on intracellular and secreted Aß. To identify regulators of sAPPα and Aß secretion, various inhibitors of intracellular trafficking were administered with DAPG. Brefeldin A (BFA) reversed DAPG-mediated changes in Aß secretion in 293sw cells, whereas golgicide A (GCA) and BFA were effective in primary astrocytes, indicating a cell type-specific regulation of the trafficking. Moreover, GCA or BFA effects on sAPPα, but not Aß, levels in primary astrocytes resembled those of ADAM10 inhibition, indicating at least partly independent trafficking pathways for sAPPα and Aß. In conclusion, DAPG might be a promising drug candidate against AD regulating ADAM10 and intracellular trafficking, but optimizing DAPG ability to cross the BBB will be needed.

Glutamate carboxypeptidase II: an amyloid peptide-degrading enzyme with physiological function in the brain.

Proteolytic processing of amyloid peptides (Aßs) is one important mechanism that controls the brain Aß level. Although several Aß-degrading enzymes were identified, evidence has suggested the presence of other peptidases. Here, we report a novel function of glutamate carboxypeptidase II (GCPII) in Aß degradation in brain, which is a peptidase involved in N-acetylaspartylglutamate cleavage, folate metabolism, and prostate tumorigenesis. Maldi-Tof/MS analysis showed that recombinant human GCPII cleaved the Aß1-40 and Aß1-42 monomers at their C-termini, producing smaller fragments, and Aß1-14 that lacked aggregation property and cellular toxicity. GCPII also degrades soluble oligomers and fibrils and can reduce the endogenous plaque size in brain sections obtained from amyloid precursor protein (APP) Swedish/presinilin (PS)-1ΔE9 transgenic mice. Overexpression of GCPII in either HEK293-APP Swedish cells or primary neurons and glial cells reduced the levels of secreted or exogenously supplemented Aßs and reduced Aß-induced neurotoxicity, suggesting the biological significance of GCPII-mediated Aß cleavage. Moreover, treatment of 8-mo-old transgenic mice for 1 mo with 2-(phosphonomethyl)-pentanedioic acid (10 mg/kg, intraperitoneally), a specific GCPII inhibitor, increased cerebral Aß content. These results suggest an important physiological role for GCPII in Aß clearance in brain and provide the evidence that dysregulation of GCPII is involved in Alzheimer's disease pathology.

Identification of peripheral inflammatory markers between normal control and Alzheimer's disease.

BACKGROUND: Multiple pathogenic factors may contribute to the pathophysiology of Alzheimer's disease (AD). Peripheral blood markers have been used to assess biochemical changes associated with AD and mild cognitive impairment (MCI) and involved in their pathophysiology. METHODS: Plasma samples and clinical data were obtained from participants in the Ansan Geriatric Study (AGE study). Plasma concentrations of four candidate biomarkers were measured in the normal control (NC), MCI, and AD group: interleukin-8 (IL-8), IL-10, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α).Body mass index (BMI), MMSE (Mini Mental State Examination), CDR(Clinical Dementia Rating) score and homocystein level were recorded with social and demographic information. RESULTS: Total of 59 subjects were randomly selected for this analysis [NC (n = 21), MCI(n = 20) and AD(n = 18)]. In demographic data, educational year was correlated with the diagnosis states (p < 0.0001). No significant differences in cardiovascular disease, BMI and use of NSAIDs were found in MCI or AD group compared with NC group, respectively. The involvement of inflammatory illness or conditions in subjects, WBC count, fibrinogen and homocystein of the three groups, but no significant differences were found in each groups. The plasma IL-8 level was lower in MCI and AD patients compared with the normal control group (respectively, p < 0.0001). The MCI and AD patients had similar MCP-1, IL-10, and TNF-α level. CONCLUSIONS: Our study suggests the existence of an independent and negative relationship between plasma IL-8 levels and functional status in MCI and AD patients.

Caspases-2 and -8 are involved in the presenilin1/gamma-secretase-dependent cleavage of amyloid precursor protein after the induction of apoptosis.

The presenilin/gamma-secretase protease cleaves many type-I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP). Previous studies have shown that apoptosis induces alterations in Abeta production in a caspase-dependent manner. Here, we report that staurosporine (STS)-induced apoptosis induces caspase-8 and/or-2-dependent gamma-secretase activation. Blocking of caspase activity with caspase-8 inhibitor z-IETD-fmk, and caspase-2 inhibitor z-VDVAD-fmk reduced Abeta production by STS in H4 cells expressing the Swedish mutant of APP (HSW) or APP-C99 (H4-C99). There was no inhibitory effect of other caspases (-1, -3, -5, -6, -9) on Abeta production by STS. This finding was further supported by evidence that siRNA transfection, depleting caspase-2 or -8 levels, lowered Abeta production in HSW and H4-C99 cells without affecting expression of APP or gamma-secretase complex. In addition, Abeta production by STS was decreased by JNK inhibitors, SP600125. These results suggest that caspase-2 and/or -8 is involved in presenilin/gamma-secretase activation and Abeta production in apoptosis.