Increase In Acetylcholinesterase Research Articles

Circadian Cholinergic Rhythms: Implications for Cholinesterase Inhibitor Therapy

Therapies for Alzheimer’s disease (AD) at present augment the deteriorating cholinergic system, are reasonably well tolerated, and are convenient, given once or twice a day. They may, however, support or oppose endogenous circadian cholinergic rhythms. Drugs with a duration of action longer than a day are at odds with the physiology of the cholinergic system, which is active during the day and quiescent at night. Sleep and the consolidation of daytime experience into memory may be disturbed. Tolerance commonly develops, substantial counterregulatory increases in acetylcholinesterase (AChE) have been measured, and brain AChE inhibition is lower than predicted. Therefore, the duration of action and timing of administration, as they relate to natural cholinergic rhythms, are factors to be considered in optimizing cholinergic AD therapeutics.

Neuronal–Glial Interactions Mediated by Interleukin-1 Enhance Neuronal Acetylcholinesterase Activity and mRNA Expression

Cholinergic dysfunction in Alzheimer's disease has been attributed to stress-induced increases in acetylcholinesterase (AChE) activity. Interleukin-1 (IL-1) is overexpressed in Alzheimer's disease, and stress-related changes in long-term potentiation, an ACh-related cerebral function, are triggered by interleukin-1. Microglial cultures (N9) synthesized and released IL-1 in response to conditioned media obtained from glutamate-treated primary neuron cultures or PC12 cells. This conditioned media contained elevated levels of secreted beta-amyloid precursor protein (sAPP). Naive PC12 cells cocultured with stimulated N9 cultures showed increased AChE activity and mRNA expression. These effects on AChE expression and activity could be blocked by either preincubating the glutamate-treated PC12 supernatants with anti-sAPP antibodies or preincubating naive PC12 cells with IL-1 receptor antagonist. These findings were confirmed in vivo; IL-1-containing pellets implanted into rat cortex also increased AChE mRNA levels. Neuronal stress in Alzheimer's disease may induce increases in AChE expression and activity through a molecular cascade that is mediated by sAPP-induced microglial activation and consequent overexpression of IL-1.

Calcineurin Enhances Acetylcholinesterase mRNA Stability during C2-C12 Muscle Cell Differentiation

Treatment of C2-C12 mouse myoblasts with the immunosuppressant drug cyclosporin A (CsA) enhances the increase in acetylcholinesterase (AChE) expression observed during skeletal muscle differentiation. The enhanced AChE expression is due primarily to increased mRNA stability because CsA treatment increases the half-life of AChE mRNA, but not the apparent transcriptional rate of the gene. Neither tacrolimus (FK506), an immunosuppressive agent with a distinct structure, nor cyclosporine H, an inactive congener of CsA, alters AChE expression. The enhanced AChE expression is associated with the muscle differentiation process, but cannot be triggered by CsA exposure before differentiation. Myoblasts and myotubes of C2-C12 cells express similar amounts of cyclophilin A and FKBP12, immunophilins known to be intracellular-binding targets for CsA and tacrolimus, respectively. However, cellular levels of calcineurin, a calcium/calmodulin-dependent phosphatase known to be the cellular target of ligand-immunophilin complexes, increase 3-fold during myogenesis. Overexpression of constitutively active calcineurin in differentiating cells reduces AChE mRNA levels and CsA antagonizes such an inhibition. Conversely, overexpression of a dominant negative calcineurin construct increases AChE mRNA levels, which are further enhanced by CsA. Thus, a CsA sensitive, calcineurin mediated pathway appears linked to differentiation-induced stabilization of AChE mRNA during myogenesis.

Biological actions of the epidermal growth factors-like domain peptides of mouse schwannoma-derived growth factor and human amphiregulin.

Several members of the epidermal growth factor (EGF) family of growth factors that contain EGF-like units at their carboxy portion have been isolated and characterized. Schwannoma-derived growth factor (SDGF) and amphiregulin (AR) are members of this family. SDGF has high sequence homology to AR, and is known to be not only a potent mitogen for astrocytes and fibroblasts but also a neurotrophic factor. We previously confirmed that the synthetic EGF-like peptides SDGF(38-80) and AR(44-84), corresponding to the EGF-like domain of mouse SDGF and human AR, respectively, formed similar disulfide bond patterns to that of EGF. In the present study, we further investigated the biological actions of these two EGF-like peptides on several cultured cell lines. We found that SDGF(38-80) and AR(44-84) have weak mitogenic activity in NIH/3T3 cells and weak binding affinity to the EGF receptor on the surface of A431 cells compared with EGF. However, SDGF(38-80) and EGF induced short neurite outgrowth in PC12 h cells, a subclone of PC12 cells, at 100 nM. In addition, a significant increase in acetylcholinesterase (AChE) activity induced by SDGF(38-80) was observed at a concentration similar range to that of EGF, which is known as a differentiation marker of these cells. The effect of AR(44-84) in PC12 h cells was weaker than those of SDGF(38-80) and EGF, but the AChE activity was significantly increased by the addition of 100 nM AR(44-84), which did not stimulate NIH/3T3 cell growth. These results also suggest that SDGF(38-80) and AR(44-84) may be effective for neuronal differentiation rather than proliferation.

Density of Choline Acetyltransferase-Immunoreactive Terminals in the Rat Dentate Gyrus after Entorhinal Cortex Lesions: A Quantitative Light Microscope Study

Lesion of the entorhinal cortex in the adult rat is a model for Alzheimer's disease and produces a marked increase in acetylcholinesterase (AChE) activity in the outer molecular layer (OML) of the dentate gyrus. This has been attributed to the sprouting of cholinergic axons terminals in response to denervation of the OML. The aim of this study was to investigate the density changes of cholinergic terminals in the OML at the light microscope level by using choline acetyltransferase (ChAT) immunohistochemistry and quantitative analysis. The results showed that between days 10 and 33 after an entorhinal cortex lesion, there was a measurable increase in the density of ChAT-positive boutons in the OML of the ipsilateral dentate gyrus (×1.2–1.6 of contralateral). However, when shrinkage of the ipsilateral OML (×0.5–0.75 of contralateral) was taken into account, the apparent increase in ChAT terminal density was entirely accounted for by shrinkage of the OML. Thus ChAT immunohistochemistry at the light microscope level provides no positive evidence for a proliferation of cholinergic terminals in the entorhinal cortex lesion model. This is in agreement with previous biochemical assays that have shown no change of total ChAT activity in the dentate gyrus after entorhinal cortex lesions.

Amyloid Precursor Protein Fragment and Acetylcholinesterase Increase with Cell Confluence and Differentiation in a Neuronal Cell Line

This study addresses the developmental regulation of amyloid precursor protein (APP) fragments comprising the amyloid-β peptide (Aβ) and the amyloid-promoting factor acetylcholinesterase (AChE) in a mouse neuronal cell line (Neuro-2a). Results indicate that a 35-kDa amyloidogenic fragment of APP and the major molecular forms of AChE (G1and G4) in Neuro-2a cells significantly increase with increasing levels of cell confluence. The foregoing molecules undergo further increases when neuroblastoma cells differentiate in the presence of dibutyryl cAMP. In contrast, a 17-kDa fragment of APP and butyrylcholinesterase were not affected by cell confluence or differentiation. These findings are the first to indicate that a selective Aβ-containing fragment of APP is subject to developmental regulation. Moreover, our data show that the 35-kDa fragment and AChE forms respond in parallel to the same developmental stimuli, i.e., cell confluence and differentiation. This points to the existence of a functional relationship between both molecules, a notion that is consistent with the potential role that has been ascribed to AChE in both APP processing and the formation of amyloid deposits in Alzheimer's brains.

Phenotype of intraadrenal ganglion neurons during postnatal development in rat

The postnatal development of intraadrenal ganglion neurons was studied in rat by using indirect immunohistochemistry and in situ hybridization. The large neuropeptide tyrosine (NPY)-expressing ganglion neurons (type I ganglion neurons) matured postnatally, with marked increases in acetylcholinesterase (AChE)-, neurofilament 10 (NF10)-, and tyrosine hydroxylase (TH)-like immunoreactivities (LIs) paralleled by increasing levels of mRNAs encoding NPY, low-affinity neurotrophin receptor (LANR), and tropomyosin kinase receptor (trk). The smaller vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) ganglion neurons (type II ganglion neurons) expressed increasing levels of VIP mRNA postnatally and also contained immunoreactive nitric oxide synthase (NOS) and its mRNA. These type II ganglion neurons appeared to be relatively mature already at postnatal day (P2) and did not express detectable levels of LANR or trk mRNAs. The cell size of both the type I and type II ganglion neurons increased about 2.5-fold postnatally. The type I ganglion neurons formed more densely packed clusters with increasing age, whereas the type II ganglion neurons were spread out in small groups or individually, mainly in the peripheral parts of the medulla, and appeared to fulfill their migration into the medulla and/or to the inner regions of the cortex early postnatally, possibly after establishing contact with their cortical targets. We suggest that the type I ganglion neurons represent sympathetic ganglion neurons of the same origin as the chromaffin cells and that they mature mainly postnatally. The development of the type II (VIP/NOS) ganglion neurons takes place earlier; however, their phenotype remains more uncertain. © 1996 Wiley-Liss, Inc.

Phenotype of intraadrenal ganglion neurons during postnatal development in rat.

The postnatal development of intraadrenal ganglion neurons was studied in rat by using indirect immunohistochemistry and in situ hybridization. The large neuropeptide tyrosine (NPY)-expressing ganglion neurons (type I ganglion neurons) matured postnatally, with marked increases in acetylcholinesterase (AChE)-, neurofilament 10 (NF10)-, and tyrosine hydroxylase (TH)-like immunoreactivities (LIs) paralleled by increasing levels of mRNAs encoding NPY, low-affinity neurotrophin receptor (LANR), and tropomyosin kinase receptor (trk). The smaller vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) ganglion neurons (type II ganglion neurons) expressed increasing levels of VIP mRNA postnatally and also contained immunoreactive nitric oxide synthase (NOS) and its mRNA. These type II ganglion neurons appeared to be relatively mature already at postnatal day (P2) and did not express detectable levels of LANR or trk mRNAs. The cell size of both the type I and type II ganglion neurons increased about 2.5-fold postnatally. The type I ganglion neurons formed more densely packed clusters with increasing age, whereas the type II ganglion neurons were spread out in small groups or individually, mainly in the peripheral parts of the medulla, and appeared to fulfill their migration into the medulla and/or to the inner regions of the cortex early postnatally, possibly after establishing contact with their cortical targets. We suggest that the type I ganglion neurons represent sympathetic ganglion neurons of the same origin as the chromaffin cells and that they mature mainly postnatally. The development of the type II (VIP/NOS) ganglion neurons takes place earlier; however, their phenotype remains more uncertain.

Infusions of pressor agents selectively attenuate depressor responses to ACh in anesthetized dogs.

In dogs anesthetized with pentobarbital sodium (30 mg/kg iv), infusions of phenylephrine (PE; 1-3 micrograms.kg-1.min-1 iv for 30 min and longer) caused sustained elevations in blood pressure and suppressed depressor responses to acetylcholine (ACh; 1 microgram/kg iv) in a dose- and time-dependent manner. The dose-response curve for ACh (0.01-100 micrograms/kg iv)-induced depressor responses was shifted to the right by approximately 80-fold after an intravenous infusion of PE (3 micrograms.kg-1.min-1) for 120 min. Similar suppression was observed when infusions of methoxamine (5 micrograms.kg-1.min-1 iv), norepinephrine (3 micrograms.kg-1.min-1 iv under blockade of beta-adrenoceptors), or angiotensin II (0.3 micrograms.kg-1.min-1 iv) were carried out. However, in dogs treated with prazosin (1 mg/kg iv) or hydralazine (1 mg/kg iv) to prevent elevations in blood pressure over the baseline level, PE (3 micrograms.kg-1.min-1 iv) failed to attenuate depressor responses to ACh. The suppression observed after PE infusion was specific to ACh-induced depressor responses; i.e., no suppression was observed on the depressor responses to other drugs, such as histamine, sodium nitroprusside, carbachol, and methacholine. Furthermore, neostigmine (bolus injection of 30 microgram/kg iv followed by an infusion of 15 micrograms.kg-1.h-1 iv) greatly diminished the suppressive effect of PE. Except for a slight increase in acetylcholinesterase (AChE) activity in renal arterial segments, activities of both AChE and butyryl-cholinesterase in plasma, erythrocytes, and pulmonary and renal arterial segments were unchanged after PE infusion. These results suggest that prolonged elevation in blood pressure and/or vasoconstriction selectively attenuates depressor responses to ACh through accelerated degradation of this material.

P-5-10 - (+)-Mirtazapine and (+)-desmethylmirtazapine levels in rat brain in relation to in vivo blockade of α 2 receptors

Cholinergic signalling in the hippocampus may contribute to the aetiology of mood regulation. Antidepressants can reverse the increase in acetylcholinesterase (AChE) activity induced by olfactory bulbectomy. The activation of nicotinic acetylcholine receptors (nAChRs) also alleviates the symptoms of depression. This study advances the development of sarsasapogenin, which interacts with cholinergic signalling and has a favourable antidepressant profile in olfactory bulbectomised (OB) rats. We examined OB-induced changes in cholinergic signalling, as well as AChE, α4-nAChR, and α7-nAChR expression in the hippocampus. The results indicate that abnormal cholinergic signalling in the hippocampus contributes to the development of depression in the OB rat model. This depression may be alleviated following treatment with sarsasapogenin.

Increase in acetylcholinesterase in the molecular layer of the dentate gyrus in the absence of septal inputs following selective granule cell lesions

Granule cell lesions cause an increase in acetylcholinesterase (AChE) staining in the molecular layer of the dentate gyrus. The source of this response was examined by combining granule cell lesions with lesions of the fornix-fimbria, thereby removing the cholinergic input from the septum to the hippocampus. The increased AChE staining was present in animals with granule cell lesions regardless of whether the fornix was lesioned or intact. The increase in AChE staining occurred without a corresponding increase in choline acetyltransferase staining. These findings suggest that an AChE-positive, but non-cholinergic, sprouting response occurred within the dentate gyrus following selective lesions of the granule cells. The source of this sprouting may be from AChE-positive hilar interneurons.

Neurite extension and increased expression of RI cyclic AMP-binding protein in ouabain-resistant neuroblastoma mutants.

Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N-18 mouse neuroblastoma cell line selected for their ouabain-resistant (ouar) property. When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N-18 cells. The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell. This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000-dalton RI cyclic AMP (cAMP)-binding protein. The growth rate, intracellular level of cAMP, and acetylcholinesterase activity of the ouar cell lines were not significantly different from those of the parental N-18 neuroblastoma cells. Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6, O2-dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and RI cAMP-binding activities. The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP-induced differentiated neuroblastoma cell suggests that these two protocols yielded different degrees of differentiation. Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells.

Changes in the cholinergic system of lymphocytes due to mitogenic stimulation

A significant increase in acetylcholinesterase (AChE) activity and muscarinic-type cholinergic receptor (mAChR) expression was demonstrated in normal human peripheral blood lymphocytes as an early response to various stimuli. AChE activity of lymphocytes from T-cell acute lymphoid leukemia patients, and that of leukemic cell lines was enhanced or increased only slightly, in good accordance with their significantly decreased response to mitogenic stimuli. The higher initial AChE activity of cultured leukemic cell lines probably reflects their permanently activated state. AChE activity and mAChR expression in these cases could only be increased by a differentiation-inducing agent. Based on these observations, the possible role of the cholinergic modulation in thymic lymphocyte maturation is assumed.

Functional reactivation of the deafferented hippocampus by embryonic septal grafts as assessed by measurements of local glucose utilization.

Transection of the septo-hippocampal connections through fimbria-fornix damage in the rat results in profound hippocampal cholinergic deafferentation, and, when applied bilaterally, leads to severe and long-lasting impairments in learning and memory. Previous studies have shown that intrahippocampal septal grafts can reestablish a new cholinergic in the initially denervated hippocampal formation and at least partly compensate for the lesion-induced learning impairments in fimbria-fornix lesioned rats. The purpose of the present study was to determine the magnitude of lesion-induced alterations in cerebral function as reflected in local glucose use measured by (14C)-2-deoxyglucose (2-DG) autoradiography, and the degree to which this index of functional activity could be normalized following reinnervation from transplants of fetal cerebral tissue from the primordial septal region. Six months after unilateral fimbria-fornix transection the rate of glucose utilization was reduced markedly throughout the ipsilateral hippocampus when compared to the intact contralateral side, while in the neocortex only the cingulate cortex showed long-lasting reductions in glucose use. Rats that received a transplant of fetal septal-diagonal band tissue at the time of fimbria-fornix transection, and were sacrificed 6 months later, displayed significantly greater glucose utilization in the ipsilateral hippocampus and cingulate cortex than was measured in these areas in rats with lesion alone. The recovery in glucose use was paralleled by a significant increase in acetylcholinesterase (AChE) staining in several areas of the ipsilateral hippocampal formation and cingulate cortex. This index of graft-induced cholinergic reinnervation was, moreover, significantly correlated with the rate of glucose use. Thus, in the fimbria-fornix transected animals the magnitude of glucose depression correlated with the extent of reduction in AChE staining, and in the grafted animals the degree of normalization of glucose use was correlated with the graft-induced increase in AChE-staining density. These results thus indicate that the 2-DG autoradiographic technique can provide a unique opportunity to map both altered functional activity in localized areas of the brain following specific lesions and the extent to which transplant-derived reinnervation of the host may induce a return to normal functional levels in the target site.

Influence of skin illumination on serum acetylcholinesterase of the rabbit.

The effects of skin illumination (continuous or discontinuous colorless or yellow filtered white light) on acetylcholinesterase levels are tested on rabbit serum. Significant increases in acetylcholinesterase are observed with continuous and discontinuous colorless filtered white light as compared to controls.

Early septal lesion: Effect on the development of the cholinergic system in rat hippocampus

The development of the cholinergic system in the rat hippocampal formation was studied following lesion of the septal region at an age of 2–4 days postnatal (i.e. the lesion was performed prior to the establishment of the septohippocampal connections). The steep increase in acetylcholinesterase (AChE) level, that under normal conditions take place during the second and the third week postnatal, was not observed in early lesioned animals, and AChE level at maturity was about 30% of control. AChE level of adult-lesioned animals was about 15% of control, suggesting an age-dependent plasticity in response to the lesion. Early deafferentation did not seem to alter the pattern of development of muscarinic binding sites as measured by specific binding of [ 3H]quinuclidinyl benzilate ([ 3H]QNB). Total [ 3H]QNB bound per hippocampus of adult, early-lesioned animals was about 70% of control, but this reduction could be accounted for by the atrophy observed in the hippocampal formation following early lesion. Binding of [ 3H]QNB per protein in early lesioned animals did not differ from normal. Thus the development and the level of muscarinic binding sites in the hippocampal formation do not seem to depend upon normal establishment of presynaptic contacts.

Role of the adenylate cyclase system in inductive acetylcholinesterase synthesis in the brain

The increase in acetylcholinesterase (ACE) activity in the rat brain after intraventricular injection of adrenalin and the dibutyryl analog of cyclic AMP was shown to be the result of inductive synthesis of the enzyme. Induction of ACE is manifested to a greater degree in the white matter of the subcortex than in the cortex. Blocking β-adrenergic receptors inhibits the stimulating action of adrenalin on ACE activity but does not alter the effect of cyclic AMP. Blocking of the α-adrenergic receptors, on the other hand, potentiates induction of synthesis of the enzyme. The effects of adrenalin and of dibutyryl-cyclic AMP are similar in direction and are mediated through β-adrenergic receptors. The increase in ACE activity after blocking of α receptors can be explained by the elimination of their inhibitory effect on β-adrenergic receptors.

Regulation of acetylcholinesterase in cultured muscle by chemical agents and electrical stimulation

Cultures of 11 day old chick embryo pectoral muscle were used to study the effects of direct electrical stimulation and neurochemicals such as acetylcholine, acetyl-β-methylcholine, tetrodotoxin, and d-tubocurarine on the acetylcholinesterase levels of muscle. The results suggest that excitation-contraction is an important factor in regulation of muscle acetylcholinesterase. Tetrodotoxin, acetylcholine and its analog acetyl-β-methylcholine increased acetylcholinesterase levels and reduced spontaneous contractions, d-tubocurarine blocked the increase in acetylcholinesterase and the decrease in spontaneous contractions caused by acetyl-β)-methylcholine. Electrical stimulation decreased acetylcholinesterase and increased muscle contractions in normal and in diisopropylfluorophosphate treated cultures. Tetrodotoxin also affected the morphology of the muscle cells, as if it adversely affected normal growth and differentiation. Electrical stimulation did not increase muscle creatine kinase.

Effects of median nerve injury on some digital tissues of the hand of the macaque (Macaca fascicularis).

Median nerve injury experiments in Macaca fascicularis showed a reciprocal decrease and increase in acetylcholinesterase (AChE) and butyrylcholinesterase (ChE) activity in the digital Meissner’s corpuscles during degeneration and regeneration. During degeneration AChE activity was absent but became abundant during early regeneration of the nerve endings and slowly declined to normal during late regeneration. In contrast, ChE activity remained unchanged during early degeneration but showed a progressive decline during early regeneration and became intense in late regeneration. Experiments involving arterial and nerve section gave results that suggest the presence of a small periarterial neural contribution to the digital sweat glands.

Binding of d-[dimethyl-14C]tubocurarine, acetylcholinesterase and proteolipids in subcellular membranes of cerebral cortex of the developing rat.

Abstract— The cerebral cortex of rats at postnatal ages of 5,15,30 and 50 days was homogenized and fractionated to separate the crude mitochondrial fraction. This fraction was osmotically shocked and the Mi fraction and subfractions were separated. The variations with age in the morphological composition of subfractions M1 0·8, Mi 1·3 and M1p were studied under the electron microscope. Because of the changes observed in the various fractions the need for such type of control is stressed.The changes in total protein and proteolipid‐protein of the fractions at different ages, as well as the acetylcholinesterase activity and the binding of d‐[dimethyl−14C]tubocurarine were studied. The results obtained were interpreted on the basis of the important morphogenetic changes that the cerebral cortex undergoes postnatally. The progressive and parallel increase in acetylcholinesterase and in the binding of d‐[dimethyl‐14C]tubocurarine to proteolipids of fraction Mi 1·3, suggest a close relationship between these two events and the development of the cholinergic synapses.