Differential effects of statins and alendronate on cholinesterases in serum and brain of rats

Acetylcholinesterase (AChE) inhibitors represent standard treatment of Alzheimer's disease. Cholesterol plays an important role in Alzheimer's disease development. Because cholesterol synthesis may be inhibited by statins or bisphosphonates, we hypothesized that these drugs might possibly have an influence on cholinesterases. Moreover, we also evaluated if the cholesterol-lowering agents that cross the blood-brain barrier (e.g. simvastatin) should be more effective than those which do not (e.g. atorvastatin). Four groups of rats were orally administered simvastatin, atorvastatin, alendronate or vehicle for seven days. Thereafter, blood samples were taken and the basal ganglia, septum, frontal cortex, and hippocampus were isolated from brains for measurement of acetylcholinesterase activity. In the blood, activities of neither acetyl- nor butyrylcholinesterase were influenced by any of the applied drugs. In the brain, no significant changes in AChE activity were observed after administration of atorvastatin. Both simvastatin and alendronate significantly suppressed the activity of AChE in the frontal cortex. In conclusion, our results confirmed the hypothesis that cholesterol-modifying drugs modulate AChE activity and it is more reasonable to use a blood-brain barrier penetrating drug.     

Novel oxoisoaporphine-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

A series of novel oxoisoaporphine-based inhibitors (10-aminoalkylamino-1-azabenzanthrone Ar-NH(CH(2))(n)NR(1)R(2)) of acetylcholinesterase (AChE) has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE) and AChE-induced β-amyloid (Aβ) aggregation. The synthetic compounds exhibited high AChE inhibitory activity with IC(50) values in the submicromolar range in most cases. Non-competitive binding mode was found for these derivatives by the graphical analysis of steady-state inhibition data. Moreover, all compounds exhibit significant inhibitory activity on AChE-induced Aβ aggregation with inhibitory potency from 54.5% to 93.5%. Finally, six out of twelve synthetic compounds were predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. The result encourages us to study this class of compounds thoroughly and systematically.     

Synthesis of derivatives of cleistopholine and their anti-acetylcholinesterase and anti-β-amyloid aggregation activity

A series of 6- and 9-substituted cleistopholine derivatives has been designed, synthesized and investigated to inhibit the aggregation of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-myloid (A β). Results showed that these synthetic compounds had excellent AChE inhibitory activity and a significant in vitro inhibitory potency toward the self-induced A β aggregation. When SH-SY5Y cells were treated with these substituted cleistopholine derivatives during they overexpressed the Swedish mutant form of human β -amyloid precursor protein (APPsw), A β 42 secretion levels were significantly reduced. According to a parallel artificial membrane permeation assay for BBB, seven out of these sixteen synthetic compounds probably could cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS).     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

mRNA expression levels of tight junction protein genes in mouse brain capillary endothelial cells highly purified by magnetic cell sorting

Tight junctions (TJs) are an important component of the blood-brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of brain capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 10⁶ cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the brain capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-brain barrier in cooperation with claudin-5.     

Structure-based design,synthesis, and evaluation of structurally rigid donepezil analogues as dual AChE and BACE-1 inhibitors

A new series of structurally rigid donepezil analogues was designed, synthesized and evaluated as potential multi-target-directed ligands (MTDLs) against neurodegenerative diseases. The investigated compounds 10–13 displayed dual AChE and BACE-1 inhibitory activities in comparison to donepezil, the FDA-approved drug. The hybrid compound 13 bearing 2-aminoquinoline scaffold exhibited potent AChE inhibition (IC₅₀ value of 14.7?nM) and BACE-1 inhibition (IC₅₀ value of 13.1?nM). Molecular modeling studies were employed to reveal potential dual binding mode of 13 to AChE and BACE-1. The effect of the investigated compounds on the viability of SH-SY5Y neuroblastoma cells and their ability to cross the blood-brain barrier (BBB) in PAMPA-BBB assay were further studied.     

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.     

A chlorodiazirine analog of pentamidine with anti-trypanosomal activity

A chlorodiazirine derivative of pentamidine was synthesized and tested for anti-trypanosomal activity using EATRO stock 164 trypanosomes in cell culture. Anti-trypanosomal activity was measured as a decrease in [3H]hypoxanthine incorporation by the organisms. The derivative, 3,3'-[1,5-pentanediylbis(oxy-4,1-phenylene)]bis(3-chloro-3H-diazir ine), at a treatment level of 0.1 microM inhibited isotope incorporation by 40-50% compared to nontreated controls. At this concentration, pentamidine inhibited incorporation only 10-15%. The derivative is a nonionic molecule with much different solubility properties than the parent compound and should readily cross the blood-brain barrier.     

Effects of duration of positive-pressure ventilation on blood-brain barrier function in premature lambs.

We have been studying the ontogeny of the blood-brain barrier function in ovine fetuses and lambs. During these studies, we have found that the duration of ventilation also influences blood-brain barrier permeability in premature lambs. Chronically instrumented hysterotomy-delivered surfactant-treated premature lambs were studied at 90% or 137 days of gestation (n = 9). Blood-brain barrier function was quantified with the blood-to-brain transfer constant K(i) to alpha-aminoisobutyric acid. Linear regression analysis was used to compare the K(i) values in the brain regions, as the dependent variable, to the duration of ventilation, as the independent variable. There were direct correlations (P < 0.05) between the K(i) values and the duration of ventilation [306 min (mean), 162-474 min (range)] in the cerebral cortex, cerebellum, medulla, caudate nucleus, hippocampus, superior colliculus, inferior colliculus, thalamus, pons, cervical spinal cord, and choroid plexus, but not in the pituitary gland. Ventilatory pressures and rates were established before the onset of the permeability studies. Calculated mean airway pressures [14 cmH(2)O (mean), 7-20 cmH(2)O (range)] from similarly studied premature lambs did not correlate with the duration of positive-pressure ventilation. We conclude that increases in the duration of positive-pressure ventilation predispose premature lambs to increases in regional blood-brain barrier permeability. These alterations in barrier function occur over relatively short time intervals (minutes to hours). In our study, these changes in permeability are most likely not attributable to changes in mean airway pressure.     

In vitro penetration of des-tyrosine1-D-phenylalanine3-beta-casomorphin across the blood-brain barrier.

The blood-brain barrier transport and metabolism of the synthetic beta-casomorphin (beta CM) derivative des-tyrosine1-D-phenylalanine3-beta-casomorphin (DT-D-Phe3-beta CM) were investigated using an in vitro model consisting of primary cultures of bovine cerebrovascular endothelial cells. DT-D-Phe3-beta CM was transported across the endothelial monolayer without significant metabolism. The endothelial permeability expressing the transport rate ranged between 1.4 and 2.2 cm x 10(-3)/min and was neither affected by luminal concentration changes (1 nM and 1 microM) nor different after luminal and abluminal administration. The metabolic inhibitor 2-desoxy-D-glucose did not affect the permeability of DT-D-Phe3-beta CM. These results suggest that DT-D-Phe3-beta CM is able to cross the blood-brain barrier by paracellular transport without using a carrier system.     

Conformational changes in proteins in vitro as a means of predicting the acute toxicities of chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Bj?rn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.     

Transgenic and antisense manipulations of impairments in cholinergic balance

Inhibitors of acetylcholinesterase (AChE) catalysis are increasingly used as insecticides as well as therapeutic and prophylactic agents. However, the long‐term consequences of their use are not yet known. To investigate this topic, we used cultured neurons and transgenic mouse pedigrees that over‐express natural variants of human AChE. Following exposure to an anti‐AChE, alternative splicing of the pre‐mRNA from the single ACHE gene produces the relatively rare AChE‐R mRNA variant in mice and in cultured cerebellar neurons. This promotes a rapid (minutes) yet long‐lasting (weeks) translocation of AChE‐R mRNA into neurites that is associated with extreme neuronal hypersensitivity to both anti‐AChEs and atropine (Meshorer et al. 2002, Science, 295 , 508‐512). In transgenic mice, over‐expression of AChE‐R causes stress‐induced irregular bursts of increased locomotor activity and failure of short‐term memory (Cohen et al. Mol. Psych., in press). Nanomolar concentrations of an antisense oligonucleotide selectively suppress AChE‐R mRNA levels in both hyperproducing cultured neurons and mouse brain. Moreover, this treatment reverses the behavioral and cognitive deficits of the mice (effect apparent for > 24 h, vs. < 45 min for tacrine). The efficacy of our antisense experiments raises the possibility of eventually using such agents to achieve AChE variant‐specific suppression, long‐lasting effect, and, presumably, fewer side‐effects than is offered by conventional anticholinesterase therapy.     

Transforming Growth Factor-β1 Upregulates the Tight Junction and P-glycoprotein of Brain Microvascular Endothelial Cells

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.     

Transport of Leucine-Enkephalin Across the Blood-Brain Barrier in the Perfused Guinea Pig Brain

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of peripheral CCK receptor blockade on feeding responses to duodenal nutrient infusions in rats

Type A cholecystokinin receptor (CCKAR) antagonists differing in blood-brain barrier permeability were used to test the hypothesis that duodenal delivery of protein, carbohydrate, and fat produces satiety in part by an essential CCK action at CCKARs located peripheral to the blood-brain barrier. Fasted rats with open gastric fistulas received devazepide (1 mg/kg iv) or A-70104 (700 nmol. kg(-1). h(-1) iv) and either a 30-min intravenous infusion of CCK-8 (10 nmol. kg(-1). h(-1)) or duodenal infusion of peptone, maltose, or Intralipid beginning 10 min before 30-min access to 15% sucrose. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide, does not. CCK-8 inhibited sham feeding by approximately 50%, and both A-70104 and devazepide abolished this response. Duodenal infusion of each of the macronutrients dose dependently inhibited sham feeding. A-70104 and devazepide attenuated inhibitory responses to each macronutrient. Thus endogenous CCK appears to act in part at CCKARs peripheral to the blood-brain barrier to inhibit food intake.     

Prevention of scopolamine-induced memory deficits by schisandrin B, an antioxidant lignan from Schisandra chinensis in mice

The preventive effect of schisandrin B (Sch B), an antioxidant ingredient of Schisandra chinensis, was studied on scopolamine-induced dementia in mouse. Scopolamine developed oxidative stress in the brain with the decreased levels of antioxidant enzymes and increased nitrite level. At the same time, a significant impairment of learning and memory occurred when evaluated by passive avoidance task (PAT) and Morris water maze (MWM) with concomitant increase of acetylcholinesterase (AChE) activity and decreased acetylcholine levels. Pre-treatment by Sch B (10, 25, 50 mg/kg) effectively prevented scopolamine-induced oxidative stress and improved behavioural tasks. Further, the scopolamine-induced increase in AChE activity was significantly suppressed and the level of acetylcholine was maintained as normal by Sch B treatment. These results suggest that Sch B have protective function against cerebral functional defects such as dementia not only by antioxidant prevention but also exerting its potent cognitive-enhancing activity through modulation of acetylcholine level.     

In Silico Studies in Probing the Role of Kinetic and Structural Effects of Different Drugs for the Reactivation of Tabun-Inhibited AChE

We have examined the reactivation mechanism of the tabun-conjugated AChE with various drugs using density functional theory (DFT) and post-Hartree-Fock methods. The electronic environments and structural features of neutral oximes (deazapralidoxime and 3-hydroxy-2-pyridinealdoxime) and charged monopyridinium oxime (2-PAM) and bispyridinium oxime (Ortho-7) are different, hence their efficacy varies towards the reactivation process of tabun-conjugated AChE. The calculated potential energy surfaces suggest that a monopyridinium reactivator is less favorable for the reactivation of tabun-inhibited AChE compared to a bis-quaternary reactivator, which substantiates the experimental study. The rate determining barrier with neutral oximes was found to be ∼2.5 kcal/mol, which was ∼5.0 kcal/mol lower than charged oxime drugs such as Ortho-7. The structural analysis of the calculated geometries suggest that the charged oximes form strong O^…H and N^…H hydrogen bonding and C-H^…π non-bonding interaction with the tabun-inhibited enzyme to stabilize the reactant complex compared to separated reactants, which influences the activation barrier. The ability of neutral drugs to cross the blood-brain barrier was also found to be superior to charged antidotes, which corroborates the available experimental observations. The calculated activation barriers support the superiority of neutral oximes for the activation of tabun-inhibited AChE compared to charged oximes. However, they lack effective interactions with their peripheral sites. Docking studies revealed that the poor binding affinity of simple neutral oxime drugs such as 3-hydroxy-2-pyridinealdoxime inside the active-site gorge of AChE was significantly augmented with the addition of neutral peripheral units compared to conventional charged peripheral sites. The newly designed oxime drug 2 appears to be an attractive candidate as efficient antidote to kinetically and structurally reactivate the tabun-inhibited enzyme.     

RAGE: a potential target for Abeta-mediated cellular perturbation in Alzheimer's disease

This review focuses on the current findings regarding interaction between amyloid beta peptide (Abeta) and receptor for advanced glycation endproducts (RAGE) and its roles in the pathogenesis of Alzheimer's disease (AD). As a ubiquitously expressed cell surface receptor, RAGE mediates the effects of Abeta on microglia, blood-brain barrier (BBB) and neurons through activating different signaling pathways. Data from autopsy brain tissues, in vitro cell cultures and transgenic mouse models suggest that Abeta-RAGE interaction exaggerates neuronal stress, accumulation of Abeta, impaired learning memory, and neuroinflammation. Blockade of RAGE protects against Abeta-mediated cellular perturbation. These findings may have an important therapeutic implication for neurodegenerative disorders relevant to AD.     

Acetylcholinesterase activation and enhanced lipid peroxidation after long-term exposure to low levels of aluminum on different mouse brain regions

Aluminum (Al), oxidative stress and impaired cholinergic functions have all been related to Alzheimer's disease (AD). The present study evaluates the effect of aluminum on acetylcholinesterase (AChE) and lipid peroxidation in the mouse brain. Mice were loaded by gavage with Al 0.1 mmol/kg/day 5 days per week during 12 weeks. The mice were divided into four groups: (1) control; (2) 10 mg/mL of citrate solution; (3) 0.1 mmol/kg of Al solution; (4) 0.1 mmol/kg of Al plus 10 mg/mL of citrate solution. AChE activity was determined in the hippocampus, striatum, cortex, hypothalamus and cerebellum and lipid peroxidation was determined in the hippocampus, striatum and cortex. An increase of AChE activity was observed in the fourth group (Al + Ci) in the hippocampus (36%), striatum (54%), cortex (44%) and hypothalamus (22%) (p<0.01). The third group (Al) presented a decrease of AChE activity in the hypothalamus (20%) and an enhancement in the striatum (27%). Lipid peroxidation, measured by TBARS (thiobarbituric acid reactive substances), was elevated in the hippocampus and cerebral cortex when compared with the control (p < 0.01). The effect of aluminum on AChE activity may be due to a direct neurotoxic effect of the metal or perhaps a disarrangement of the plasmatic membrane caused by increased lipid peroxidation.     

Design,synthesis and biological evaluation of 2-acetyl-5-O-(amino-alkyl)phenol derivatives as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetyl-5-O-(amino-alkyl)phenol derivatives was designed, synthesized and evaluated as multi-function inhibitors for the treatment of Alzheimer’s disease (AD). The results revealed that compound TM-3 indicated selective AChE inhibitory potency (eeAChE, IC₅₀ = 0.69 μM, selective index (SI) = 32.7). Both kinetic analysis of AChE inhibition and molecular modeling study suggested that TM-3 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. And TM-3 was also a highly selective MAO-B inhibitor (IC₅₀ = 6.8 μM). Moreover, TM-3 could act as antioxidant (ORAC value was 1.5eq) and neuroprotectant, as well as a selective metal chelating agent. More interestingly, compound TM-3 could cross the blood-brain barrier (BBB) in vitro and abided by Lipinski’s rule of five. Therefore, compound TM-3, a promising multi-targeted active molecule, offers an attractive starting point for further lead optimization in the drug-discovery process against AD.