Specific Acetylcholinesterase Research Articles

Combined Donepezil with Astaxanthin via Nanostructured Lipid Carriers Effective Delivery to Brain for Alzheimer's Disease in Rat Model.

Donepezil (DPL), a specific acetylcholinesterase inhibitor, is used as a first-line treatment to improve cognitive deficits in Alzheimer's disease (AD) and it might have a disease modifying effect. Astaxanthin (AST) is a natural potent antioxidant with neuroprotective, anti-amyloidogenic, anti-apoptotic, and anti-inflammatory effects. This study aimed to prepare nanostructured lipid carriers (NLCs) co-loaded with donepezil and astaxanthin (DPL/AST-NLCs) and evaluate their in vivo efficacy in an AD-like rat model 30 days after daily intranasal administration. DPL/AST-NLCs were prepared using a hot high-shear homogenization technique, in vitro examined for their physicochemical parameters and in vivo evaluated. AD induction in rats was performed by aluminum chloride. The cortex and hippocampus were isolated from the brain of rats for biochemical testing and histopathological examination. DPL/AST-NLCs showed z-average diameter 149.9 ± 3.21 nm, polydispersity index 0.224 ± 0.017, zeta potential -33.7 ± 4.71 mV, entrapment efficiency 81.25 ±1.98% (donepezil) and 93.85 ±1.75% (astaxanthin), in vitro sustained release of both donepezil and astaxanthin for 24 h, spherical morphology by transmission electron microscopy, and they were stable at 4-8 ± 2°C for six months. Differential scanning calorimetry revealed that donepezil and astaxanthin were molecularly dispersed in the NLC matrix in an amorphous state. The DPL/AST-NLC-treated rats showed significantly lower levels of nuclear factor-kappa B, malondialdehyde, β-site amyloid precursor protein cleaving enzyme-1, caspase-3, amyloid beta (Aβ1‑42), and acetylcholinesterase, and significantly higher levels of glutathione and acetylcholine in the cortex and hippocampus than the AD-like untreated rats and that treated with donepezil-NLCs. DPL/AST-NLCs showed significantly higher anti-amyloidogenic, antioxidant, anti-acetylcholinesterase, anti-inflammatory, and anti-apoptotic effects, resulting in significant improvement in the cortical and hippocampal histopathology. Nose-to-brain delivery of DPL/AST-NLCs is a promising strategy for the management of AD.

Diversity of Endophytic Fungi in Huperzia serrata and Their Acetylcholinesterase Inhibitory Activity

Huperzia serrata is a Huperzine A (HupA)-producing herb. HupA is a potent, reversible, highly specific, centrally active and selective acetylcholinesterase inhibitor, and is commonly used to improve Alzheimer’s disease.At present, H. serrata resources have been overexploited and their artificial cultivation has lagged behind the need, so the market demand cannot be met. In this study, the diversity of the endophytic fungi in H. serrata was studied by high-throughput sequencing and traditional culture methods. Furthermore, the ability of the isolated endophytic fungi to produce acetylcholinesterase-inhibiting activity was evaluated. The results related to the diversity of fungi between high-throughput sequencing and traditional culture methods were compared. With high-throughput sequencing, five phyla, 22 classes, 55 orders, 120 families, and 178 genera of fungi were detected in Hubei Province, and five phyla, 22 classes, 54 orders, 124 families, and 196 genera were detected in Fujian Province. After cultivation with traditional culture methods, two phyla, three classes, five orders, six families, and six genera were detected in Hubei, and one phylum, three classes, five orders, five families, and six genera in Fujian. The endophytic fungi of H. serrata are highly diverse, and the results of high-throughput sequencing comprehensively reflect their community compositions. When the acetylcholinesterase-inhibiting activity of the isolated and cultured endophytic fungi was determined, five and three endophytic fungi from Hubei and Fujian Provinces, respectively, had good inhibitory activity (inhibition rate > 50%). Most of them were separated from leaf tissue. The strain HBR-1 and strain FJL-5 had the strongest inhibitory effects on AChE, with inhibition rates of 72.34% and 60.54%, respectively. Although only a proportion of the endophytic fungi was isolated with traditional culture methods, metabolites with broad potential applications can be isolated from these fungal cultures. The combination of high-throughput sequencing and traditional culture methods can be used to isolate and purify endophytic fungi in a targeted manner, gain many endophytic fungi, and enrich the endophytic fungi resource library.

Screening and identification of acetylcholinesterase inhibitors from Terminalia chebula fruits based on ultrafiltration and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Traditional Chinese medicines (TCMs) are one of the most important sources of numerous lead compounds in drug discovery. However, rapid screening and identification of bioactive compounds from complex medicinal plants remain a technical challenge. In this work, a method based on ultrafiltration (UF) and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS/MS) was developed to rapidly screen and identify specific acetylcholinesterase (AChE) inhibitors from Terminalia chebula fruits. The water extract of T. chebula fruits was subjected to column chromatography on D101 macroporous resin with increasing proportions of ethanol as the eluent to produce five fractions. Fraction E2 (30% ethanol eluent) exhibited the strongest AChE inhibitory activity and was incubated with AChE. After incubation, ultrafiltration and dissociation, 17 compounds including eight gallotannins, seven ellagitannins and two other compounds in fraction E2 were screened out and their molecular structures were identified by MS/MS spectra. Subsequently, the commercially available standards of chebulagic acid, corilagin, chebulanin and ellagic acid were used to verify the compound identification, AChE inhibitory assay in vitro and molecular docking. The study revealed that chebulanin, chebulagic acid and corilagin were responsible for AChE inhibitory activities with IC50 values of 0.35 ± 0.02 mM, 0.42 ± 0.03 mM and 0.72 ± 0.03 mM, respectively. Chebulagic acid, corilagin, chebulanin, and ellagic acid could fit well into the binding pocket of the model with binding energy values ranging from −8.7 to −6.4 kcal/mol. In conclusion, the results suggested that the developed method could be a rapid, effective, convenient and high-throughput screening approach for AChE inhibitors from TCMs.

Acetylcholinesterase, Specific Acetylcholinesterase and Total Protein Concentrations in the Brain Regions of Broiler Chickens Fed Dietary Monosodium Glutamate

The study was carried out to examine the effect of varied levels of dietary monosodium glutamate on acetylcholinesterase, specific acetylcholinesterase and total protein concentrations in the brain regions of broiler chickens. Three hundred (300) day – old unsexed Abor – acre chickens were randomly assigned to diets: A, B, C, D, E and F containing 0.00, 0.25, 0.50, 0.75, 1.00 and 1.25 g/kg MSG respectively. Each treatment was replicated 5 times with 10 birds per replicate. The birds were fed ad – libitum and provided with clean water for 8 weeks (56 days) after which 2 birds per replicates were slaughtered. The brains were removed, dissected into different regions comprising of the olfactory lobe, pineal body, optic lobe, cerebellum and the medulla oblongata. The different parts of the brain were homogenized to determine the acetylcholinesterase and total protein which were also used in the assessment of the specific acetylcholinesterase of the brain. No significant differences were observed in the acetylcholinesterase activity of the olfactory lobe, pineal body, optic lobe, cerebellum except for the medulla. Likewise, the dietary monosodium glutamate did not influence the activities of the total protein and specific acetylcholinesterase of the olfactory lobe portion of the brain. The dietary monosodium glutamate exerted significant effects on the total protein of other brain parts studied and which invariably resulted in significant changes in the specific acetylcholinesterase of the optic lobe, cerebellum and medulla except for the optic lobe. This study revealed that monosodium glutamate added to broilers diet above 0.75 g/kg significantly altered the concentration of the brain acetylcholinesterase, total protein and specific acetylcholinesterase thereby impaired brain functions.

Involvement of Acetylcholine Receptors in Cholinergic Pathway-Mediated Protection Against Autoimmune Diabetes.

Type I diabetes (T1D) is a T cell-driven autoimmune disease that results in the killing of pancreatic β-cells and, consequently, loss of insulin production. Using the multiple low-dose streptozotocin (MLD-STZ) model of experimental autoimmune diabetes, we previously reported that pretreatment with a specific acetylcholinesterase inhibitor (AChEI), paraoxon, prevented the development of hyperglycemia in C57BL/6 mice. This correlated with an inhibition of T cell infiltration into the pancreatic islets and a reduction in pro-inflammatory cytokines. The cholinergic anti-inflammatory pathway utilizes nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) expressed on a variety of cell types. In this study, we carried out a comparative analysis of the effect of specific antagonists of nAChRs or mAChRs on the development of autoimmune diabetes. Co-administration of mecamylamine, a non-selective antagonist of nAChRs maintained the protective effect of AChEI on the development of hyperglycemia. In contrast, co-administration of atropine, a non-selective antagonist of mAChRs, mitigated AChEI-mediated protection. Mice pretreated with mecamylamine had an improved response in glucose tolerance test (GTT) than mice pretreated with atropine. These differential effects of nAChR and mAChR antagonists correlated with the extent of islet cell infiltration and with the structure and functionality of the β-cells. Taken together, our data suggest that mAChRs are essential for the protective effect of cholinergic stimulation in autoimmune diabetes.

Role of acetylcholine receptors in cholinergic pathway-mediated protection against autoimmune diabetes

Abstract Type I diabetes (T1D) is a T cell-driven autoimmune disease that results in the killing of pancreatic β-cells and, consequently, loss of insulin production. Using the multiple low-dose streptozotocin (MLD-STZ) model of experimental autoimmune diabetes, we previously reported that pretreatment with a specific acetylcholinesterase inhibitor (AChEI), paraoxon, prevented the development of hyperglycemia in C57BL/6 mice. This correlated with an inhibition of T cell infiltration into the pancreatic islets and a reduction in proinflammatory cytokines. The cholinergic anti-inflammatory pathway utilizes nicotinic and muscarinic acetycholine receptors (AChR) expressed on a variety of cell types. In this study, we carried out a comparative analysis on the effect of specific antagonists of the nicotinic- or muscarinic-AChR on the development of autoimmune diabetes. Co-administration of nicotinic-AChR antagonist (mecamylamine) maintained the protective effect of AChEI on the development of hyperglycemia. In contrast, co-administration with muscarinic-AChR antagonist (atropine) mitigated AChEI-mediated protection. Mice pretreated with mecamylamine had an improved response in glucose tolerance test than mice pretreated with atropine. These findings correlated with the extent of islet cell infiltration and with the structure and functionality of the β-cells. Taken together, our data suggest that muscarinic receptors are essential for the protective effect of cholinergic stimulation on autoimmune diabetes.

Analysis of cholinergic pathway-induced alterations at the intestinal mucosa and their association with enhanced resistance to lethal bacterial infection

Abstract The mucosal innate immune system represents the first line of defense against microorganisms invading the intestinal tract. Increasing evidence suggests that mucosal immune defenses are tightly regulated by immunological and neural pathways. We previously demonstrated that activation of the cholinergic pathway through the administration of a highly specific acetylcholinesterase inhibitor (AChEI), paraoxon, confers resistance to an oral infection by virulent S. typhimurium. This protection is mediated by enhanced innate anti-microbial mucosal defenses in the GI tract. In the present study, we investigated the involvement of different immune cell populations in the GI tract in the modulation of AChEI-mediated anti-Salmonella resistance. Morphological evaluation of intestinal tissue from control- or AChEI-treated mice revealed a significant thickening of the mucin layer and increased B and T lymphocytic infiltration into intestinal mucosa in the AChEI group. Multi-color flow cytometric analysis of iliac intraepithelial lymphocytes (IELs) showed that cholinergic stimulation increased the percentage of CD3+CD8+gamma/delta TCR+ cells. No significant alterations were observed in lamina propria cells. Our findings indicate that cholinergic pathway is able to modulate not only mucin-secreting cells but more importantly gamma/delta T cells, the cell population known to be involved in tissue homeostasis and repair as well as surveillance of the epithelial barrier.

Physiologic changes in acetylcholinesterase, specific acetylcholinesterase and total protein concentrations in the brain of domestic fowl (Gallus domesticus) cocks fed dietary monosodium glutamate

Physiologic changes in acetylcholinesterase, specific acetylcholinesterase and total protein concentrations in the brain of domestic fowl (Gallus domesticus) cocks fed dietary monosodium glutamate

Cholinergic Activation Enhances Resistance to Oral Salmonella Infection by Modulating Innate Immune Defense Mechanisms at the Intestinal Barrier.

Inflammation is a crucial defense mechanism that protects the body from the devastating effects of invading pathogens. However, an unrestrained inflammatory reaction may result in systemic manifestations with dire consequences to the host. The extent of activation of the inflammatory response is tightly regulated through immunological and neural pathways. Previously, we demonstrated that cholinergic stimulation confers enhanced protection in experimental animals orally infected with virulent Salmonella enterica serovar Typhimurium. In this study, we investigated the mechanism by which this enhanced protection takes place. Cholinergic stimulation was induced by a 3-week pretreatment with paraoxon, a highly specific acetylcholinesterase (AChE) inhibitor. This treatment enhanced host survival following oral-route infection and this correlated with significantly reduced bacterial load in systemic target organs. Enhanced protection was not due to increased gut motility or rapid bacterial clearance from the gastrointestinal tract. Moreover, protection against bacterial infection was not evident when the animals were infected systemically, suggesting that acetylcholine-mediated protective effect was mostly confined to the gut mucosal tissue. In vivo imaging demonstrated a more localized infection and delay in bacterial dissemination into systemic organs in mice pretreated with paraoxon. Morphological analysis of the small intestine (ileum) showed that AChE inhibition induced the degranulation of goblet cells and Paneth cells, two specialized secretory cells involved in innate immunity. Our findings demonstrate a crucial pathway between neural and immune systems that acts at the mucosal interface to protect the host against oral pathogens.

Cholinergic Stimulation Prevents the Development of Autoimmune Diabetes: Evidence for the Modulation of Th17 Effector Cells via an IFNγ-Dependent Mechanism.

Type I diabetes (T1D) results from T cell-mediated damage of pancreatic β-cells and loss of insulin production. The cholinergic anti-inflammatory pathway represents a physiological link connecting the central nervous and immune systems via vagus nerve, and functions to control the release of proinflammatory cytokines. Using the multiple low-dose streptozotocin (MLD-STZ) model to induce experimental autoimmune diabetes, we investigated the potential of regulating the development of hyperglycemia through administration of paraoxon, a highly specific acetylcholinesterase inhibitor (AChEI). We demonstrate that pretreatment with paraoxon prevented hyperglycemia in STZ-treated C57BL/6 mice. This correlated with a reduction in T cell infiltration into pancreatic islets and preservation of the structure and functionality of β-cells. Gene expression analysis of pancreatic tissue revealed that increased peripheral cholinergic activity prevented STZ-mediated loss of insulin production, this being associated with a reduction in IL-1β, IL-6, and IL-17 proinflammatory cytokines. Intracellular cytokine analysis in splenic T cells demonstrated that inhibition of AChE led to a shift in STZ-induced immune response from a predominantly disease-causing IL-17-expressing Th17 cells to IFNγ-positive Th1 cells. Consistent with this conclusion, inhibition of AChE failed to prevent STZ-induced hyperglycemia in IFNγ-deficient mice. Our results provide mechanistic evidence for the prevention of murine T1D by inhibition of AChE and suggest a promising strategy for modulating disease severity.

PH-dependent hydrolysis of acetylcholine: Consequences for non-neuronal acetylcholine

Acetylcholine is inactivated by acetylcholinesterase and butyrylcholinesterase and thereby its cellular signalling is stopped. One distinguishing difference between the neuronal and non-neuronal cholinergic system is the high expression level of the esterase activity within the former and a considerably lower level within the latter system. Thus, any situation which limits the activity of both esterases will affect the non-neuronal cholinergic system to a much greater extent than the neuronal one. Both esterases are pH-dependent with an optimum at pH above 7, whereas at pH values below 6 particularly the specific acetylcholinesterase is more or less inactive. Thus, acetylcholine is prevented from hydrolysis at such low pH values. The pH of the surface of the human skin is around 5 and therefore non-neuronal acetylcholine released from keratinocytes can be detected in a non-invasive manner. Several clinical conditions like metabolic acidosis, inflammation, fracture-related haematomas, cardiac ischemia and malignant tumours are associated with local or systemic pH values below 7. Thus, the present article describes some consequences of an impaired inactivation of extracellular non-neuronal acetylcholine.

Tools for Resistance Monitoring in Oriental Fruit Moth (Lepidoptera: Tortricidae) and First Assessment in Brazilian Populations

In southern Brazilian apple (Malus spp.) orchards, predominantly organophosphates are used to control the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae), but control failures often occur. Therefore the susceptibility of three C. molesta Brazilian populations was investigated to five insecticides of different groups and modes of action, in comparison with a susceptible laboratory strain mass reared in southern France for >10 yr. At the same time, comparative biochemical and genetic analysis were performed, assessing the activities of the detoxification enzymatic systems and sequencing a gene of insecticide molecular target to find out markers associated with resistance. The three Brazilian populations were significantly resistant to chlorpyrifos ethyl compared with the reference strain. One of the field populations that had been frequently exposed to deltamethrin treatments showed significant decreasing susceptibility to this compound, whereas none of the three populations had loss of susceptibility to tebufenozide and thiacloprid compared with the reference strain. All three populations had slight but significant increases of glutathione transferase and carboxylesterases activities and significant decrease of specific acetylcholinesterase activities compared with the reference. Only the most resistant population to chlorpyriphos exhibited a significantly higher mixed function oxidase activity than the reference. The acetylcholinesterase of females was significantly less inhibited by carbaryl in the Brazilian populations than in the reference strain (1.7-2.5-fold), and this difference was not expressed in the male moth. However, no mutation in the MACE locus was detected. These biological and molecular characterizations of adaptive response to insecticides in C. molesta provide tools for early detection of insecticide resistance in field populations of this pest.

ChemInform Abstract: Carbamate Ester Prodrugs of Dopaminergic Compounds: Synthesis, Stability, and Bioconversion.

Various carbamic acid esters (CAE) of a new class of dopaminergic drugs, 5-substituted 8-chloro-7-hydroxy-3-methyl-2,3,4,5- tetrahydro-1 H-3-benzazepines, were synthesized and evaluated as prodrug forms with the aim of protecting the parent phenols against first-pass metabolism following oral administration. Monosubstituted CAE were found to be highly unstable at pH 7.4 and 37 degrees C, the half-lives of hydrolysis being between 4 and 40 min. Plasma from various species catalyzed the hydrolysis of the carbamates. N,N-Disubstituted carbamates, on the other hand, were stable both in buffer and plasma solutions. They showed a very potent inhibition of butyrylcholinesterase (EC 3.1.1.8), but were less potent inhibitors of the specific erythrocyte acetylcholinesterase (EC 3.1.1.17). In vitro incubations of an N,N-dimethylsubstituted carbamate ester (10) with liver microsomes from mouse and rat showed an appreciable formation of the parent phenolic compound. This bioconversion is suggested to occur via an initial cytochrome P-450-catalyzed hydroxylation to give an N-hydroxymethyl derivative which spontaneously decomposes to the N-monomethylcarbamate. It is concluded that N,N-disubstituted carbamate esters may be potentially useful prodrugs for the 7-hydroxy-3-benzazepines, whereas N-monosubstituted carbamates appear to be too chemically and enzymatically labile.

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate iodomethylate N-beta-(acetoxyethyl)piperidinium ("piperidylcholine") and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic analysis parameters - activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholine esterase substrate - N-beta-(acetoxyethyl)piperidinium.

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: Next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 μg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48 h following the final and highest (400 μg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100 μg to >50% at 400 μg, and peaking 1.5 h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5 h after the final 400 μg dose (5.47 ± 2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C 18 reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.

Lymph heart in chick - somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Rivastigmine is a potent inhibitor of acetyl- and butyrylcholinesterase in Alzheimer's plaques and tangles

Acetylcholinesterase and butyrylcholinesterase activities emerge in association with plaques and tangles in Alzheimer's disease. These pathological cholinesterases, with altered properties, are suggested to participate in formation of plaques. The present experiment assessed the ability of rivastigmine, a clinically utilized agent that inhibits acetylcholinesterase and butyrylcholinesterase activities, to inhibit cholinesterases in plaques and tangles. Cortical sections from cases of Alzheimer's disease were processed using cholinesterase histochemistry in the presence or absence of rivastigmine. Optical densities of stained sections were utilized as a measure of inhibition. The potency of rivastigmine was compared with those of other specific inhibitors. Optimum staining for cholinesterases in neurons and axons was obtained at pH 8.0. Cholinesterases in plaques, tangles and glia were stained best at pH 6.8. Butyrylcholinesterase-positive plaques were more numerous than acetylcholinesterase-positive plaques. Rivastigmine inhibited acetylcholinesterase in all positive structures in a dose-dependent manner (10 −6–10 −4 M). However, even at the highest concentration, faint activity remained. In contrast, rivastigmine resulted in complete inhibition of butyrylcholinesterase in all structures at 10 −5 M. Rivastigmine was equipotent to the specific acetylcholinesterase inhibitor BW284C51 and more potent than the butyrylcholinesterase inhibitors iso-OMPA and ethopropazine. In conclusion, rivastigmine is a potent inhibitor of acetylcholinesterase and a more potent inhibitor of butyrylcholinesterase in plaques and tangles. Unlike other cholinesterase inhibitors tested, rivastigmine inhibited cholinesterases in normal and pathological structures with the same potency. Thus, at the therapeutic concentrations used, rivastigmine is likely to result in inhibition of pathological cholinesterases, with the potential of interfering with the disease process.

Functional Manipulations of Acetylcholinesterase Splice Variants Highlight Alternative Splicing Contributions to Murine Neocortical Development

Proliferation and differentiation of mammalian central nervous system progenitor cells involve concertedly controlled transcriptional and alternative splicing modulations. Searching for the developmental implications of this programming, we manipulated specific acetylcholinesterase (AChE) splice variants in the embryonic mouse brain. In wild type mice, 'synaptic' AChE-S appeared in migrating neurons, whereas the C-terminus cleaved off the stress-induced AChE-R variant associated with migratory radial glial fibers. Antisense suppression of AChE-R reduced neuronal migration, allowing increased proliferation of progenitor cells. In contrast, transgenic overexpression of AChE-R was ineffective, whereas transgenic excess of enzymatically active AChE-S or inactive AChE-Sin suppressed progenitors proliferation alone or both proliferation and neuronal migration, respectively. Our findings attribute to alternative splicing events an interactive major role in neocortical development.

Induction of Corneal Lesion and Nerve Fiber Sprouting by Neonatal Capsaicin Application Depends on the Dose of the Drug and Survival Time after Treatment

The effects of capsaicin on the rat cornea and its NsAchE (non specific acetylcholinesterase)-positive nerve fibers were investigated after long and short survival periods following subcutaneous (s.c) injection of the drug. Sixteen rats were injected once s.c with capsaicin on postnatal day 2 at a dose of 50 mg/kg. Age-matched rats were injected only vehicle, and served as control (n = 16). After 4 (n = 6), 8 (n = 6) and 12 (n = 4) mos., both sides of the corneas were examined under a binocular microscope to look for corneal abnormalities. Immediately after the enucleation, bilateral corneas were excised with a thin scleral margin and their ciliary body and iris were removed in DPBS solution. Then, they were fixed in a 4% paraformaldehyde solution containing 8% sucrose for 1 h at 4 degrees C, and processed for staining by the NsAchE method. For comparison, 15 rats were injected 3 times with capsaicin (total dose: 150 mg/kg) at a dose of 50 mg/kg on days 1, 2 and 3 after birth. Age-matched rats (n = 16) were injected vehicle as controls. Five (n = 4), 17 (n = 4), 45 (n = 3) and 75 (n = 4) days later, their corneas were similarly handled as described above. Corneal lesions and sprouting of the NsAchE-positive subepithelial nerve fibers appeared 4 mos. after the treatment with capsaicin (50 mg/kg). In particular, all the treated corneas (8/8) at 12 mos. showed corneal abnormalities. Contrary to the results from the single injection of capsaicin, corneal changes had already appeared on day 17 after treatment with capsaicin at a high dose (150 mg/kg). These data reinforce the suggestion that yielding of corneal lesions is closely associated with the longer survival time at lower doses and with the short survival time at a high dose of the drug. Further, the relationship between sprouting of corneal nerve fibers and corneal wounds was discussed on the basis of the previous reports.

Sargaquinoic acid promotes neurite outgrowth via protein kinase A and MAP kinases-mediated signaling pathways in PC12D cells

We previously isolated a nerve growth factor (NGF)-dependent neurite outgrowth promoting substance MC14 (sargaquinoic acid) from a marine brown alga, Sargassum macrocarpum. In the present study, the NGF-potentiating activity of MC14 to neural differentiation of PC12D cells was investigated in detail. The treatment of cells with 3μg/ml MC14 in the presence of 1.25–100ng/ml NGF markedly enhanced the proportion of neurite-bearing cells compared with the NGF-only controls. In addition, MC14 significantly elevated the NGF-induced specific acetylcholinesterase (AchE) activity in PC12D cells, suggesting that MC14 could morphologically and biochemically promote the differentiation of PC12D cells. The mechanism of action of MC14 was further investigated by pharmacological inhibition of several intracellular signaling molecules. Results indicated that the neurite outgrowth promoting activity of MC14 was almost completely blocked by 10μM PD98059, suggesting that a TrkA-dependent MAP kinases-mediated signaling pathway may play a crucial role in modulating the effect of MC14. Besides, the MC14-enhanced neurite outgrowth was substantially suppressed by the pretreatment with 10ng/ml protein kinase A (PKA) inhibitor, demonstrating that the adenylate cyclase–PKA signaling cascade was also involved in the action of MC14. In contrast, a PKC inhibitor chelerythrine chloride did not inhibit the neurite outgrowth promoting activity of MC14. Altogether, these results demonstrate that MC14 enhances the neurite outgrowth by cooperating at least two separated signaling pathways, a TrkA–MAP kinases pathway and an adenylate cyclase–PKA pathway, in PC12D cells.