Enzyme assays are important for studying enzyme-mediated biochemical reactions and for clinical diagnosis and drug development. The technique of an immobilized enzyme microreactor (IMER) integrated with capillary electrophoresis (CE) has been frequently utilized in online enzyme assays. However, the traditional approaches for IMER-CE enzyme analysis have some defects such as low loading capacity and poor stability. Herein, metal-organic frameworks (MOFs), which have enormous potential in the fields of enzyme immobilization and capillary electrochromatographic (CEC) separation, were first explored as novel support materials with good enzyme immobilization performance and stationary phases with excellent separation abilities to construct an integrated MOFs-IMER-CEC microanalysis system for a high-efficiency online enzyme assay. As a proof-of-concept demonstration, acetylcholinesterase (AChE) was immobilized on a densely packed UiO-66-NH(2) nanocrystal coating on a capillary inner surface with abundant intercrystalline mesoporosity and was employed as a highly effective and robust IMER for CEC-integrated online enzyme analysis. The excellent separation performance of the UiO-66-NH(2)-modified capillary was verified by high-efficiency separation of three types of neutral, acidic, and basic compounds. The Michaelis-Menten constant and enzyme inhibition kinetics of UiO-66-NH(2)-IMER were systematically assessed, exhibiting distinct advantages such as remarkably increased enzyme loadability, superior affinity for substrates, and greatly improved stability and repeatability compared to CE-integrated IMERs prepared by the traditional covalent bonding method. Furthermore, the developed method was successfully utilized for detecting organophosphorus pesticides in leguminous vegetable samples, demonstrating its strong practicality. The study not only proposed a novel support material and construction strategy for a high-performance microchannel-based IMER but also can be widely used in bioanalysis and biosensing research.
As a by-product in the sericulture industry, mulberry branches are not currently utilized effectively. Jialing 20 is an artificial triploids mulberry that widely cultivated in southwest China. In this study, the chemical composition of the Jialing 20 mulberry branch extract (MBE) was first analyzed by UPLC-MS/MS, and 42 components, including alkaloids, flavonoids, and coumarins, were obtained. Then, the antioxidant activities, hypoglycemic effect, Alzheimer's disease inhibition, and cardiovascular protection of MBE were also evaluated in vitro. The IC(50) values for the scavenging DPPH and ABTS radicals were, respectively, 31.23 +/- 0.57 microg/mL and 8.88 +/- 0.36 microg/mL (IC(50) values of positive Vc were, respectively, 4.41 +/- 0.19 microg/mL and 8.79 +/- 0.41 microg/mL). The IC(50) value for inhibiting alpha-glucosidase was 1.90 +/- 0.05 microg/mL (IC(50) value of positive acarbose was 0.03 microg/mL). The IC(50) values for inhibiting acetylcholinesterase and butyrylcholinesterase were, respectively, 179.47 +/- 0.38 microg/mL and 101.82 +/- 3.37 microg/mL (IC(50) values of positive berberine were, respectively, 1.27 +/- 0.03 microg/mL and 57.41 +/- 0.21 microg/mL). MBE (10 microg/mL and 40 microg/mL) significantly increased the survival rate of oxidized low-density lipoprotein- (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) and significantly decreased the intracellular reactive oxygen species. These results suggest that the extracts of Jialing 20 mulberry branches could be used as a functional food additive.
Multifunctional carbamate-type acetylcholinesterase (AChE) inhibitors with anti-amyloidogenic properties like phenserine are potential therapeutic agents for Alzheimer's disease (AD). We reported here the design of new carbamates using pharmacophore model strategy to modulate both cholinesterase and amyloidogenesis. A five-feature pharmacophore model was generated based on 25 carbamate-type training set compounds. (-)-Meptazinol carbamates that superimposed well upon the model were designed and synthesized, which exhibited nanomolar AChE inhibitory potency and good anti-amyloidogenic properties in in vitro test. The phenylcarbamate 43 was highly potent (IC50 31.6 nM) and slightly selective for AChE, and showed low acute toxicity. In enzyme kinetics assay, 43 exhibited uncompetitive inhibition and reacted by pseudo-irreversible mechanism. 43 also showed amyloid-beta (Abeta) lowering effects (51.9% decrease of Abeta42) superior to phenserine (31% decrease of total Abeta) in SH-SY5Y-APP695 cells at 50 microM. The dual actions of 43 on cholinergic and amyloidogenic pathways indicated potential uses as symptomatic and disease-modifying agents.
AIMS: To investigate whether Meserine, a novel phenylcarbamate derivative of (-)-meptazinol, possesses beneficial activities against cholinergic deficiency and amyloidogenesis, the two major pathological characteristics of Alzheimer's disease (AD). METHODS: Ellman's assay and Morris water maze were used to detect acetylcholinesterase (AChE) activity and evaluate spatial learning and memory ability, respectively. Both high content screening and Western blotting were carried out to detect beta-amyloid precursor protein (APP), while RT-PCR and ELISA were conducted to detect APP-mRNA and beta-amyloid peptide (Abeta). RESULTS: In scopolamine-induced dementia mice, Meserine (1 mg/kg, i.p.) significantly ameliorated spatial learning and memory deficits, which was consistent with its in vitro inhibitory ability against AChE (recombinant human AChE, IC50 = 274 +/- 49 nM). Furthermore, Meserine (7.5 mg/kg) injected intraperitoneally once daily for 3 weeks lowered APP level by 28% and Abeta42 level by 42% in APP/PS1 transgenic mouse cerebrum. This APP modulation action might be posttranscriptional, as Meserine reduced APP by about 30% in SH-SY5Y-APP695 cells but did not alter APP-mRNA level. And both APP and Abeta42 lowering action of Meserine maintained longer than that of rivastigmine. CONCLUSION: Meserine executes dual actions against cholinergic deficiency and amyloidogenesis and provides a promising lead compound for symptomatic and modifying therapy of AD.
Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progressive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-beta aggregation inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive amelioration. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two "water bridges" located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose-response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100ng/kg, comparable with the effect of a reference drug Huperzine A at 1mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics.
Dipeptidyl peptidase IV (DPP-IV) degrades the incretin hormone glucagon-like peptide 1 (GLP-1). Small molecule DPP-IV inhibitors have been used as treatments for type 2 diabetes to improve glucose tolerance. However, each of the marketed small molecule drugs has its own limitation in terms of efficacy and side effects. To search for an alternative strategy of inhibiting DPP-IV activity, we generated a panel of tight binding inhibitory mouse monoclonal antibodies (mAbs) against rat DPP-IV. When tested in vitro, these mAbs partially inhibited the GLP-1 cleavage activity of purified enzyme and rat plasma. To understand the partial inhibition, we solved the co-crystal structure of one of the mAb Fabs (Ab1) in complex with rat DPP-IV. Although Ab1 does not bind at the active site, it partially blocks the side opening, which prevents the large substrates such as GLP-1 from accessing the active site, but not small molecules such as sitagliptin. When Ab1 was tested in vivo, it reduced plasma glucose and increased plasma GLP-1 concentration during an oral glucose tolerance test in rats. Together, we demonstrated the feasibility of using mAbs to inhibit DPP-IV activity and to improve glucose tolerance in a diabetic rat model.
Title: Evaluation of a new substrate for measurement of serum PON arylesterase activity Mu X, Yu N, Wang C, Zou X, Abulimite Z, Xia Z Ref: Talanta, 88:711, 2012 : PubMed
It was found that the hydrolysis of 9-(4-chlorophenyloxycarbonyl)-10-methylacridinium triflate (CPOCMA) could be catalyzed by recombinant human PON1. Based on this property, the CPOCMA was evaluated as a substrate for serum PON activity assay. The apparent K(m) value of a serum sample for the substrate was determined as 85nmol/L, close to the K(m) value (83nmol/L) of rHuPON1. The interferences by other esterase such as acetylcholinerase and lipases were investigated. The NaCl and CaCl(2) as PON activity enhancers were able to improve the specific signal, respectively. The rHuPON1 in presence of CaCl(2) showed at least 7.8 times selectivity over acetylcholinerase and lipases. By comparing with the UV methods based on phenyl acetate and diazinon, respectively, the proposed chemiluminescent method was validated with 30 serum samples. The method based on CPOCMA allows reliable, cost-saving, and specific determination in a buffer of physiological pH.
Title: Memory defect induced by beta-amyloid plus glutamate receptor agonist is alleviated by catalpol and donepezil through different mechanisms Xia Z, Zhang R, Wu P, Hu Y Ref: Brain Research, 1441:27, 2012 : PubMed
Our previous studies demonstrate that a non-cholinesterase inhibitor (AChEI) compound catalpol, purified from a traditional Chinese medicinal herb Rehmannia glutinosa, could improve the symptoms and pathological changes in animal and cellular models of memory related neurodegenerative diseases. In this study, we compared catalpol with the most commonly used AChEI donepezil in respect to their mechanism of action on the neurodegenerative changes in an animal model induced by beta-amyloid (Abeta) plus glutamate receptor agonist. It was found that the model mice showed significant deficit in the learning ability and memory in Y maze avoidance test, and meanwhile both donepezil and catalpol greatly improve the learning ability and memory after 2 to 3 months' administration. At the selected doses, donepezil only partially raised the declined brain muscarinic acetylcholine receptor (M receptor) density and choline acetyltransferase (ChAT) activity resulting in these levels still lower than normal control, while catalpol completely retrieved these two parameters. ELISA revealed that catalpol, instead of donepezil, possessed the capability of elevating the declined brain BDNF level of the animal model. The ELISA results on the BDNF protein level was confirmed by quantitative RT-PCR measurement of BDNF mRNA in Abeta(2)(5)(-)(3)(5)-treated primary culture of forebrain neurons. In combination with our previous work, we think the neuroprotective effects of donepezil and catalpol are mediated through different mechanisms. Since BDNF has been proved to be an important intrinsic factor in protecting neurodegenerative diseases, catalpol may be a hopefully effective compound against neurodegenerative changes induced by Abeta and glutamate receptor agonist.
Title: Smilagenin attenuates beta amyloid (25-35)-induced degeneration of neuronal cells via stimulating the gene expression of brain-derived neurotrophic factor Zhang R, Wang Z, Howson PA, Xia Z, Zhou S, Wu E, Hu Y Ref: Neuroscience, 210:275, 2012 : PubMed
The development of drugs that attenuate neurodegeneration is important for the treatment of Alzheimer's disease (AD). We previously found that smilagenin (SMI), a steroidal sapogenin from traditional Chinese medicinal herbs improves memory in animal models, is neither a cholinesterase inhibitor nor a glutamate receptor antagonist, but can significantly elevate the declined muscarinic receptor (M receptor) density. In this article, to clarify whether SMI represents a new approach for treating neurodegeneration disease, we first demonstrate that SMI pretreatment significantly attenuates the neurodegenerative changes induced by beta amyloid 25-35 (Abeta(25-35)) in cultured rat cortical neurons, including decreased cholinergic neuron number, shortened neurite outgrowth length, and declined M receptor density. Brain-derived neurotrophic factor (BDNF) protein levels in the culture medium were also decreased by Abeta(25-35) and significantly elevated by SMI. Parallel experiments revealed that when the trk receptors were inhibited by K252a or the action of BDNF was inhibited by a neutralizing anti-BDNF antibody, the effects of SMI on the Abeta(25-35)-induced neurodegeneration in rat cortical neurons were almost completely abolished. In the all-trans retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells, the BDNF transcription rate measured by a nuclear run-on assay was significantly suppressed by Abeta(25-35) and elevated by SMI, but the BDNF degradation rate measured by half-life determination was unchanged by Abeta(25-35) and SMI. Transcript analysis of the SH-SY5Y cells using quantitative RT-PCR (qRT-PCR) showed that the IV and VI transcripts of BDNF mRNA were significantly decreased by Abeta(25-35) and elevated by SMI. Taken together, we conclude that SMI attenuates Abeta(25-35)-induced neurodegeneration in cultured rat cortical neurons and SH-SY5Y cells mainly through stimulating BDNF mRNA transcription implicating that SMI may represent a novel therapeutic strategy for AD.
The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(-)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC(50) values of 9.63uM (for ZLA) and 8.64uM (for ZLB), and prevent AChE-induced amyloid-beta (Abeta) aggregation with IC(50) values of 49.1uM (for ZLA) and 55.3uM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Abeta aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD.
Title: Regulation of M1-receptor mRNA stability by smilagenin and its significance in improving memory of aged rats Hu Y, Wang Z, Zhang R, Wu P, Xia Z, Orsi A, Rees D Ref: Neurobiology of Aging, 31:1010, 2010 : PubMed
The purpose of this work is to study the effect of smilagenin on the mRNA stability of muscarinic receptor subtype 1 (M(1); m1 mRNA) in aged rat brains and its significance in improving memory. The Y-maze avoidance task showed that oral administration of smilagenin significantly improved spatial memory performance in aged rats. Mechanistic studies showed that smilagenin was neither a ligand of the M receptors nor a cholinesterase inhibitor, while radioligand binding assays revealed that smilagenin significantly increased the M(1)-receptor density. The increase of M(1)-receptor density correlated with memory improvement. Real-time polymerase chain reaction (RT-PCR) revealed that the m1 mRNA in m1 gene-transfected CHO cells increased significantly, and the average half-life of m1 mRNA was approximately doubled by smilagenin treatment. These results suggest that smilagenin improves memory of aged rats at least partially by increasing the stability of m1 mRNA. However since the ChAT activity in the cortex of aged rats was also elevated by smilagenin, it cannot be excluded that the increase of intrinsic acetylcholine excretion also plays a role in the memory-improvement effect of smilagenin.
We have investigated four approximately 1.6-Mb microduplications and 55 smaller 350-680-kb microduplications at 15q13.2-q13.3 involving the CHRNA7 gene that were detected by clinical microarray analysis. Applying high-resolution array-CGH, we mapped all 118 chromosomal breakpoints of these microduplications. We also sequenced 26 small microduplication breakpoints that were clustering at hotspots of nonallelic homologous recombination (NAHR). All four large microduplications likely arose by NAHR between BP4 and BP5 LCRs, and 54 small microduplications arose by NAHR between two CHRNA7-LCR copies. We identified two classes of approximately 1.6-Mb microduplications and five classes of small microduplications differing in duplication size, and show that they duplicate the entire CHRNA7. We propose that size differences among small microduplications result from preexisting heterogeneity of the common BP4-BP5 inversion. Clinical data and family histories of 11 patients with small microduplications involving CHRNA7 suggest that these microduplications might be associated with developmental delay/mental retardation, muscular hypotonia, and a variety of neuropsychiatric disorders. However, we conclude that these microduplications and their associated potential for increased dosage of the CHRNA7-encoded alpha 7 subunit of nicotinic acetylcholine receptors are of uncertain clinical significance at present. Nevertheless, if they prove to have a pathological effects, their high frequency could make them a common risk factor for many neurobehavioral disorders.
Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.
Title: Peripheral cholinoceptor antagonist anisodamine counteracts cholinergic adverse effects and facilitates cognitive amelioration of rivastigmine Zhang WW, Xu ZP, Cui YY, Wang H, Song MK, Li J, Shao BY, Xia Z, Chen HZ Ref: J Neural Transm (Vienna), 116:1643, 2009 : PubMed
Rivastigmine is a potent acetyl- and butyrylcholinesterase inhibitor widely used for cognitive improvement in Alzheimer's disease (AD) therapy. However, dose-limiting adverse effects restrict its tolerability and clinical outcomes. This study explored new combined therapy, in which peripheral cholinergic adverse effects and central cognitive amelioration of rivastigmine were differentiated by a peripheral cholinoceptor antagonist anisodamine. The results demonstrated that rivastigmine (0.75 and 2.0 mg/kg) could significantly reverse the scopolamine-induced cognitive deficit in mice through passive avoidance test. Nevertheless, a high dose of rivastigmine (3.25 mg/kg) would compromise cognitive amelioration and produce obvious adverse effects, including hypersalivation, intestinal hyperperistalsis and muscle cramp. Interestingly, concomitant administration of anisodamine (10 mg/kg) effectively counteracted both the muscarinergic and nicotinergic adverse effects, while facilitating cognitive amelioration of rivastigmine (3.25 mg/kg). These findings provide an insight into the feasibility of combined therapy with cholinesterase inhibitors and peripheral cholinoceptor antagonists for the treatment of AD.
Bis-(-)-nor-meptazinols (bis-(-)-nor-MEPs) 5 were designed and synthesized by connecting two (-)-nor-MEP monomers with alkylene linkers of different lengths via the secondary amino groups. Their acetylcholinesterase (AChE) inhibitory activities were more greatly influenced by the length of the alkylene chain than butyrylcholinesterase (BChE) inhibition. The most potent nonamethylene-tethered dimer 5h exhibited low-nanomolar IC 50 values for both ChEs, having a 10 000-fold and 1500-fold increase in inhibition of AChE and BChE compared with (-)-MEP. Molecular docking elucidated that 5h simultaneously bound to the catalytic and peripheral sites in AChE via hydrophobic interactions with Trp86 and Trp286. In comparison, it folded in the large aliphatic cavity of BChE because of the absence of peripheral site and the enlargement of the active site. Furthermore, 5h and 5i markedly prevented the AChE-induced Abeta aggregation with IC 50 values of 16.6 and 5.8 microM, similar to that of propidium (IC 50 = 12.8 microM), which suggests promising disease-modifying agents for the treatment of AD patients.
Title: Association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning Sun P, Zhang Z, Wu F, Wan J, Jin X, Xia Z Ref: Front Med China, 1:320, 2007 : PubMed
The aim of this study was to explore the association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning (CBP). A case-control study of 268 patients with CBP and 268 healthy workers matched by age and sex, all of whom were occupationally exposed to benzene, was conducted. The single nucleotide polymorphisms (SNPs, rs2854451, rs3738047, rs2234922 and rs1051741) of EPHX1 gene and the SNP (rs751141) of EPHX2 gene were tested by the TaqMan PCR method. In the subjects carrying the genotype of EPHX1 rs3738047 GG, the risk of CBP was decreased in the individuals simultaneously carrying EPHX1 rs2234922 G (P = 0.02). Alternatively, in the subjects carrying the genotype of EPHX1 rs2234922 AA, the risk of CBP was increased in the individuals simultaneously carrying the allele of EPHX2 rs751141A (P = 0.03). It was also found that there were potential interactions between alcohol consumption and the polymorphism of EPHX1 rs1051741 (chiH (2) = 5.28, P = 0.02) or rs2234922 (chiH (2) = 6.71, P = 0.01). Compared to individuals with EPHX1 rs1051741 CC or rs2234922 AA genotype in the drinkers, the risk of CBP in those carrying genotypes of EPHX1 rs1051741 CT+TT or rs2234922 AG+GG was decreased, respectively (P = 0.04, P < 0.01). Haplotype analysis of polymorphisms in EPHX1 showed that the risk of CBP was increased in the subjects with haplotype 2 (rs2854451-A, rs3738047-G, rs2234922-A, rs1051741-C) or haplotype 4 (rs2854451-G, rs3738047-A, rs2234922-G, rs1051741-T), but decreased in those with haplotype 6 (rs2854451-G, rs3738047-G, rs2234922-G, rs1051741-T) or haplotype 10 (rs2854451-A, rs3738047-A, rs2234922-G, rs1051741-T), respectively. Logistic regression analysis revealed that smoking might play a role in modifying the risk of CBP (OR = 0.313, 95% CI: 0.123-0.794, P = 0.015). The genetic polymorphism in EPHX1 may be associated with the risk of CBP in the Chinese occupational population and further research is needed for the association between the genetic polymorphism in EPHX2 and the susceptibility to CBP.
Title: A new approach to the pharmacological regulation of memory: Sarsasapogenin improves memory by elevating the low muscarinic acetylcholine receptor density in brains of memory-deficit rat models Hu Y, Xia Z, Sun Q, Orsi A, Rees D Ref: Brain Research, 1060:26, 2005 : PubMed
The purpose of this paper is to study the basic pharmacological action of sarsasapogenin, a sapogenin from the Chinese medicinal herb Rhizoma Anemarrhenae, (abbreviated as ZMS in this paper), on learning ability and memory of three animal models: aged rats and two neurodegeneration models produced either by single unilateral injection of beta-amyloid 1-40 (Abeta1-40) plus ibotenic acid (Ibot A) or by bilateral injection of Ibot A alone into nucleus basalis magnocellularis. Y-maze test and step-through test revealed that learning ability and memory were impaired in the three models and were improved by oral administration of ZMS. ZMS did not inhibit acetylcholinesterase nor did it occupy the binding sites of muscarinic acetylcholine receptor (M receptor), hence it is neither an cholinesterase inhibitor nor an agonist or antagonist of M receptors. On the other hand, the densities of total M receptor and its M1 subtype in the brain of the three models were significantly lower than control rats, and ZMS significantly raised the densities of total M receptors and its M1 subtype. Linear regression revealed significant correlation between the learning ability/memory and the density of either total M receptor or its M1 subtype. Autoradiographic study with 3H-pirenzipine showed that the M1 subtype density was significantly lowered in cortex, hippocampus and striatum of aged rats, and ZMS could reverse these changes towards normal control level. Interestingly, the M1 receptor density after ZMS administration only approached but did not exceed that of normal young control rats. Therefore, ZMS seems to represent a new approach to the pharmacological regulation of learning and memory and appears to be not simply palliative but may modify the progression of the disease.
Title: Chlorpyrifos induces apoptosis in rat cortical neurons that is regulated by a balance between p38 and ERK/JNK MAP kinases Caughlan A, Newhouse K, Namgung U, Xia Z Ref: Toxicol Sci, 78:125, 2004 : PubMed
Chlorpyrifos, an acetylcholinesterase (AChE) inhibitor, is a widely used organophosphate pesticide. Recent concern has focused on its neurotoxicity that is not attributable to AChE inhibition. Here, we report that chlorpyrifos and chlorpyrifos-oxon, but not 3,5,6-trichloro-2-pyridinol (TCP; the breakdown product of chlorpyrifos and chlorpyrifos-oxon), induce apoptosis in primary cortical neurons cultured from embryonic day 17 or newborn rats. It is generally agreed that chlorpyrifos-oxon is approximately three orders of magnitude more potent than chlorpyrifos in inhibition of brain acetylcholinesterase activity. However, our data demonstrate that chlorpyrifos-oxon is only slightly more potent than chlorpyrifos in inducing apoptosis. This indicates that chlorpyrifos-induced apoptosis may occur independently of AChE inhibition, although AChE activity was not measured in this study. Furthermore, chlorpyrifos activates the ERK1/2 and p38 MAP kinases. Surprisingly, blocking ERK1/2 activation by the MEK inhibitor SL327 caused a small but statistically significant inhibition of apoptosis, while blocking p38 with SB202190 significantly accelerated apoptosis induced by chlorpyrifos. This suggests a pro- and anti-apoptotic role for ERK1/2 and p38, respectively. Although chlorpyrifos did not stimulate total JNK activity, it caused a sustained activation of a sub-pool of JNK in the nucleus and stimulated phosphorylation of c-Jun, a downstream target of JNK. Transient expression of a dominant negative c-Jun mutant inhibited chlorpyrifos-induced apoptosis, suggesting a role for JNK and JNK-mediated transcription in this cell death. Together, our data suggest apoptosis as a novel toxic endpoint of chlorpyrifos neurotoxicity in the brain that may be independent of AChE inhibition. Furthermore, activation of the ERK1/2 and JNK MAP kinases contributes to, while activation of the p38 MAP kinase counteracts chlorpyrifos-induced apoptosis in cortical neurons.
