Release Of Acetylcholinesterase Research Articles

A Novel Peptide Driving Neurodegeneration Appears Exclusively Linked to the α7 Nicotinic Acetylcholine Receptor.

T14, a 14mer peptide, is significantly increased in the pre-symptomatic Alzheimer's disease brain, and growing evidence implies its pivotal role in neurodegeneration. Here, we explore the subsequent intracellular events following binding of T14 to its target α7 nicotinic acetylcholine receptor (nAChR). Specifically, we test how various experimental manipulations of PC12 cells impact T14-induced functional outcomes. Three preparations were compared: (i) undifferentiated vs. NGF-differentiated cells; (ii) cells transfected with an overexpression of the target α7 nAChR vs. wild type cells; (iii) cells transfected with a mutant α7 nAChR containing a mutation in the G protein-binding cluster, vs. cells transfected with an overexpression of the target α7 nAChR, in three functional assays - calcium influx, cell viability, and acetylcholinesterase release. NGF-differentiated PC12 cells were less sensitive than undifferentiated cells to the concentration-dependent T14 treatment, in all the functional assays performed. The overexpression of α7 nAChR in PC12 cells promoted enhanced calcium influx when compared with the wild type PC12 cells. The α7345-348A mutation effectively abolished the T14-triggered responses across all the readouts observed. The close relationship between T14 and the α7 nAChR was further evidenced in the more physiological preparation of ex vivo rat brain, where T30 increased α7 nAChR mRNA, and finally in human brain post-mortem, where levels of T14 and α7 nAChR exhibited a strong correlation, reflecting the progression of neurodegeneration. Taken together these data would make it hard to account for T14 binding to any other receptor, and thus interception at this binding site would make a very attractive and remarkably specific therapeutic strategy.

Antagonism of a key peptide 'T14' driving neurodegeneration: Evaluation of a next generation therapeutic

T14, a 14mer peptide derived from the C-terminus of acetylcholinesterase (AChE) is a signalling molecule that could drive neurodegeneration via the alpha 7 nicotinic acetylcholine receptor. Its levels increase as Alzheimer’s pathology progresses; however, a cyclic variant of the compound, NBP14, can block the effects of the endogenous linear counterpart in-vitro, ex vivo, and in vivo. Here, we explore the antagonistic potential of two 6mer peptides, NBP6A and NBP6B. These are smaller linear versions of NBP14, designed to be more effective by modifying the amino acid residues to enhance receptor blockade alongside other relevant solubility parameters. The peptides were tested in-vitro in PC12 cells on three parameters, calcium influx, cell viability, and AChE release, and ex vivo using voltage sensitive dye imaging (VSDI) in rat brain slices. Neither NBP6A nor NBP6B applied alone had any effect. In PC12 cells, NBP6B was identified as the more potent molecule since it demonstrated more effective blockade of T14 action on calcium influx, cell viability, and AChE release. NBP6B was then further evaluated using VSDI, where it proved twice as potent as NBP14 in blocking the action of T14. The improved effect of NBP6B in blocking the actions of T14, combined with its smaller size suggests that this variant could have even greater therapeutic potential than its original cyclic compound, for treating neurodegenerative disorders.

Construction of Liquid Crystal-Based Sensors Using Enzyme-Linked Dual-Functional Nucleic Acid on Magnetic Beads.

The development of liquid crystal (LC)-based sensors with superior performances such as high portability, excellent stability, great convenience, and remarkable sensitivity is highly demanded. This work proposes a new strategy for constructing the LC-based sensor using enzyme-linked dual-functional nucleic acid (d-FNA) on magnetic beads (MBs). The detection of kanamycin (KA) is demonstrated as a model. Acetylcholinesterase (AChE) is assembled onto the KA aptamer-modified MBs with a d-FNA strand that consists of an AChE aptamer and the complementary sequence of a KA aptamer. As the specific recognition of KA by its aptamer triggers the release of AChE from the MBs, the myristoylcholine (Myr) solution after incubation with the MBs causes the black image of the LCs due to the formation of the Myr monolayer at the aqueous/LC interface. Otherwise, in the absence of KA, AChE is still decorated on the MBs and causes the hydrolysis of Myr. Therefore, a bright image of LCs is obtained. The detection of KA is successfully achieved with a lower detection limit of 48.1 pg/mL. In addition, a thin polydimethylsiloxane (PDMS) layer-coated glass and a portable optical device are used to improve the stability and portability of the LC-based sensor to advance potential commercial applications. Furthermore, the detection of KA in milk with a portable device is demonstrated, showing the potential of the proposed enzyme-linked LC-based sensor.

AChE as a spark in the Alzheimer's blaze - Antagonizing effect of a cyclized variant.

The amyloid precursor protein (APP), presenilin 1 (PS1), amyloid beta (Aβ), and GSK3 are the effectors, which are significantly associated with progression of Alzheimer's Disease (AD) and its symptoms. A significant protein, acetylcholinesterase (AChE) becomes dysfunctional as a result of cholinergic neuronal loss in AD pathology. However, certain associated peptides potentiate the release of primary neuropathological hallmarks, i.e., senile plaque and neurofibrillary tangles (NFTs), by modulating the alpha 7 acetylcholinesterase receptor (α7nAChR). The AChE variants, T30 and T14 have also been found to be elevated in AD patients and mimic the toxic actions of pathological events in patients. The manuscript discusses the significance of AChE inhibitors in AD therapeutics, by indicating the disastrous role of molecular alterations and elevation of AChE, accompanied with the downstream effects instigated by the peptide, supported by clinical evidence and investigations. The cyclized variant of AChE peptide, NBP14 has been identified as a novel candidate that reverses the harmful effects of T30, T14 and Aβ, mainly calcium influx, cell viability and AChE release. The review aims to grab the attention of neuro-researchers towards the significance of triggering effectors in propagating AD and role of AChE in regulating them, which can potentially ace the development of reliable therapeutic candidates, similar to NBP14, to mitigate neurodegeneration.

Antagonising a novel toxin “T14” in Alzheimer’s disease: Comparison of receptor blocker versus antibody effects in vitro

A 14mer peptide, T14, is a possible signaling molecule driving neurodegeneration. Its levels are doubled in the Alzheimer brain, but its effects can be blocked at the target alpha-7 receptor by a cyclised variant, ‘NBP14′, which has beneficial effects, in a transgenic mouse model, on the behavioral and histochemical profile. Since the antagonism of T14 has evident therapeutic potential, we explore here an alternative method of preventing its action by comparing the efficacy of NBP14 with a proprietorial polyclonal antibody against T14, ‘Ab-19′, at inhibiting three distinct effects of the peptide in PC12 cells: calcium influx, cell viability and compensatory acetylcholinesterase (AChE) release. None of these three parameters was affected by either blocking agent when applied alone. However, both NBP14 and the Ab-19 exhibited a dose-dependent profile against the actions of T14 in all three scenarios: the least sensitive effect observed was in the lower dose range, for both the antibody and the receptor blocker, in antagonizing T14-triggered release of AChE: this parameter is interpreted as indirect compensation for the T14-induced compromise of cell viability, triggered by the enhanced influx of calcium through the initial binding of the peptide to an allosteric site on the alpha-7 receptor. As such, it is the most delayed and indirect index of T14 action and thus the least relatively impacted by lowest doses of either NBP14 or Ab-19. In all three scenarios however the effects of T14 are successfully offset by either agent and thus offer two potentially very different therapies against Alzheimer’s disease.

Sub-chronic toxicity of broflanilide on the nervous system of zebrafish (Danio rerio)

ABSTRACT The new insecticide broflanilide, which possesses high insecticidal activity against agricultural pests, acts on the γ-aminobutyric acid receptor in the insect nervous system. At present, few studies assessed its effect on the nervous system of zebrafish, especially its sub-chronic toxicity on the zebrafish brain. In this study, the sub-chronic toxicity of broflanilide on the zebrafish brain was assessed. After 21 days of exposure at 36.3 μg/L, broflanilide caused oxidative damage to the zebrafish brain, increased the levels of reactive oxygen species and malondialdehyde, and inhibited the activities of superoxide dismutase, catalase, and glutathione peroxidase. Overall, these changes result in brain cell apoptosis, and inhibited growth and development. Moreover, broflanilide affects the release of acetylcholinesterase, γ-aminobutyric acid, 5-hydroxy tryptamine, and dopamine as well as the expression of related genes in the brain, leading to abnormal zebrafish behaviour. These results corroborate the sub-chronic toxicity of broflanilide on zebrafish brain, which helps the further understanding of the potential environmental risk of broflanilide.

A CASE OF CATASTROPHIC MAGNESIUM OVERDOSE

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Magnesium toxicity can occur due to decreased excretion or overconsumption and is rare in the general population. Early-onset symptoms of toxicity are nausea, flushing, weakness, and urinary retention. However, severe toxicity and its management is not well-described. We present a case of magnesium overdose with intractable hypotension. CASE PRESENTATION: A 34-year-old male presented to the emergency department after he was found unresponsive in a restaurant in the presence of empty bottles of magnesium supplements and ibuprofen. He was hypotensive, hypothermic, and emergently intubated for airway protection. His serum magnesium level was 11.7mg/dl. He was treated with gastric decontamination, intravenous calcium and admitted to the intensive care unit. Continuous Renal Replacement Therapy (CRRT) was initiated and led to a reduction in the measured serum magnesium concentration. Despite aggressive volume administration and multiple vasopressors, he had refractory shock and severe acidosis. An echocardiogram revealed adequate cardiac function; hence, he was not a candidate for Extracorporeal Membranous Oxygenation (ECMO). A trial of hydrocortisone and methylene blue also yielded no benefit to his distributive shock. His hospital course was further complicated by abdominal compartment syndrome requiring a bedside exploratory laparotomy, aspiration pneumonia, acute respiratory distress syndrome, and disseminated intravascular coagulation. Ultimately, his family elected to transition to comfort care, and the patient passed away on hospital day 4. DISCUSSION: Magnesium competitively blocks the entry of calcium into presynaptic terminals of smooth muscle cells, inhibiting the release of acetylcholinesterase, causing smooth muscle relaxation. In the setting of an overdose, this translates to refractory vasoplegia. Magnesium also alters the polarization of the cytoplasmic membrane in cardiac myocytes, causing lengthening of the action potential, thus predisposing to arrhythmias. While iatrogenic magnesium toxicity has been reported at levels of 3-5mg/dL, a level of >10mg/dL is uncommon. There may be a dose-dependent refractoriness that has not been identified in the literature so far. Treatment of severe magnesium toxicity requires urgent dialysis in conjunction with IV calcium which acts as an antagonist. If there is catecholamine resistance, methylene blue may be used to restore systemic vascular resistance. CONCLUSIONS: Severe magnesium toxicity can present with shock & acidosis. Despite dialysis and aggressive resuscitation, the vasoplegia can be refractory and lead to fatal complications, including respiratory collapse and cardiac arrest. Even with early recognition and treatment, the mortality can be high, and further studies are needed to improve outcomes. REFERENCE #1: Ajib FA, Childress JM. Magnesium Toxicity. [Updated 2021 Jan 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. REFERENCE #2: Cavell GF, Bryant C, Jheeta S. Iatrogenic magnesium toxicity following intravenous infusion of magnesium sulfate: risks and strategies for prevention. BMJ Case Rep. 2015;2015:bcr2015209499. Published 2015 Jul 31. doi:10.1136/bcr-2015-209499 REFERENCE #3: Pietro Delva, Magnesium and cardiac arrhythmias, Molecular Aspects of Medicine, Volume 24, Issues 1–3, 2003, Pages 53-62, ISSN 0098-2997, https://doi.org/10.1016/S0098-2997(02)00091-2. DISCLOSURES: No relevant relationships by Ayesha Azmeen, source=Web Response No relevant relationships by Daniel Condit, source=Web Response No relevant relationships by Angad Deengar, source=Web Response No relevant relationships by Prashant Grover, source=Web Response No relevant relationships by Samantha McPeck, source=Web Response No relevant relationships by Evan Nadler, source=Web Response No relevant relationships by Erin Rosenberg, source=Web Response No relevant relationships by Sharad Sathyan, source=Web Response

AChE mRNA expression as a possible novel biomarker for the diagnosis of coronary artery disease and Alzheimer’s disease, and its association with oxidative stress

Oxidative metabolic reactions and their by products have played a role in coronary artery disease (CAD) and Alzheimer’s disease (AD) pathogenesis. This study was carried out on 28 patients with AD, 21 patients with CAD, and 28 healthy as control. Oxidative stress biomarkers and acetylcholinesterase (AChE) activity were assayed in plasma. mRNA expression of AChE was investigated in leukocytes of patients with CAD and AD. Thus, Alzheimer’s and coronary artery patients were observed that the protein carbonyl levels and mRNA expression of AChE were increased (p<.05, p<.01, respectively). The plasma total thiol levels were decreased compared to the control group (p<.05). There was a significant relationship between amyloid β (Aβ) accumulation and oxidative stress, cholinergic gene expression. AChE gene expression and protein oxidation were increased in patients with AD and CAD. These results suggest that increased release of AChE from cells produces neurotoxic β-amyloid plaques and may cause neurodegenerative diseases.

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.

Additive Toxicity of β-Amyloid by a Novel Bioactive Peptide In Vitro: Possible Implications for Alzheimer’s Disease

Backgroundβ-amyloid is regarded as a significant factor in Alzheimer’s disease: but inefficient therapies based on this rationale suggests that additional signalling molecules or intermediary mechanisms must be involved in the actual initiation of the characteristic degeneration of neurons. One clue could be that acetylcholinesterase, also present in amyloid plaques, is aberrant in peripheral tissues such as blood and adrenal medulla that can be implicated in Alzheimer’s disease. The aim of this study was to assess the bioactivity of a fragment of acetylcholinesterase responsible for its non-enzymatic functions, a thirty amino acid peptide (“T30”) which has homologies with β-amyloid.MethodsCell viability was measured by sulforhodamine B assay and also lactate dehydrogenase assay: meanwhile, changes in the status of living cells was monitored by measuring release of acetylcholinesterase in cell perfusates using the Ellman reagent.FindingsT30 peptide and β-amyloid each have toxic effects on PC12 cells, comparable to hydrogen peroxide. However only the two peptides selectively then evoke a subsequent, enhanced release in acetylcholinesterase that could only be derived from the extant cells. Moreover, unlike hydrogen peroxide, the T30 peptide selectively shifted a sub-threshold dose of β-amyloid to a toxic effect, which also resulted in a comparable enhanced release of acetylcholinesterase.InterpretationThis is the first study comparing directly the bioactivity of β-amyloid with a peptide derived from acetylcholinesterase: the similarity in action suggests that the sequence homology between the two compounds might have a functional and/or pathological relevance. The subsequent enhanced release of acetylcholinesterase from the extant cells could reflect a primary ‘compensatory’ response of cells prone to degeneration, paradoxically providing further availability of the toxic C-terminal peptide to modulate the potency of β-amyloid. Such a cycle of events may provide new insights into the mechanism of continuing selective cell loss in Alzheimer’s disease and related degenerative disorders.

Characterisation of acetylcholinesterase release from neuronal cells

Although acetylcholinesterase (AChE) is primarily a hydrolytic enzyme, metabolising the neurotransmitter acetylcholine in cholinergic synapses, it also has some non-catalytic functions in the brain which are far less well characterised. AChE was shown to be secreted or shed from the neuronal cell surface like several other membrane proteins, such as the amyloid precursor protein (APP). Since AChE does not possess a transmembrane domain, its anchorage in the membrane is established via the Proline Rich Membrane Anchor (PRiMA), a transmembrane protein. Both the subunit oligomerisation and membrane anchor of AChE are shared by a related enzyme, butyrylcholinesterase (BChE), the physiological function of which in the brain is unclear. In this work, we have assayed the relative activities of AChE and BChE in membrane fractions and culture medium of three different neuronal cell lines, namely the neuroblastoma cell lines SH-SY5Y and NB7 and the mouse basal forebrain cell line SN56. In an effort to understand the shedding process of AChE, we have used several pharmacological treatments, which showed that it is likely to be mediated in part by an EDTA- and batimastat-sensitive, but GM6001-insensitive metalloprotease, with the possible additional involvement of a thiol isomerase. Cellular release of AChE by SH-SY5Y is significantly enhanced by the muscarinic acetylcholine receptor (mAChR) agonists carbachol or muscarine, with the effect of carbachol blocked by the mAChR antagonist atropine. AChE has been implicated in the pathogenesis of Alzheimer’s disease and it has been shown that it accelerates formation and increases toxicity of amyloid fibrils, which have been closely linked to the pathology of AD. In light of this, greater understanding of AChE and BChE physiology may also benefit AD research.

Changes of calcium channel mRNA, protein and current in NG108-15 cells after cell differentiation

Based on the characteristics of differentiated NG108-15 cells (cell membrane excitability, acetylcholine release, and activities of choline acetyltransferase and acetylcholinesterase), NG108-15 cells are extensively used to explore neuronal functions as a cholinergic cell line. In the present study, differentiation-induced alterations of voltage-gated Ca2+ channel mRNA, protein, and current were investigated in the NG108-15 cells. Real-time PCR, Western blot, and whole-cell patch-clamp data showed that differentiation caused mRNA, protein, and ion current changes of all Ca2+ channel subunits. However, the changes of mRNA, protein, and ion current are inconsistent in all Ca2+ channel subunits. Especially, P/Q- and R-type Ca2+ channel proteins do not form the functional P/Q- and R-type Ca2+ channels even if the mRNA and protein of P/Q- and R-type Ca2+ channels can be detected in NG108-15 cells. These results indicate that differentiation can modulate gene transcription, protein translation, and post-translation of the Ca2+ channels to induce the alteration of the Ca2+ ion currents in NG108-15 cells. From these data, we understand that combining real-time PCR, Western blot, and patch-clamp techniques can comprehensively unveil the modulation of the Ca2+ channels.

Release of acetylcholinesterase (AChE) from β-amyloid plaques assemblies improves the spatial memory impairments in APP-transgenic mice

The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid-β-peptide (Aβ). Amyloid deposits contain “chaperone molecules” which play critical roles in amyloid formation and toxicity. In the present work, we test an analog of hyperforin (IDN 5706) which releases the AChE from both the Aβ fibrils and the AChE-Aβ burdens in transgenic mice. Hyperforin is an acylphloroglucinol compound isolated from Hypericum perforatum (St. John's Wort), which is able to prevent the Aβ-induced spatial memory impairments and Aβ neurotoxicity. Altogether this gathered evidence indicates the important role of AChE in the neurotoxicity of Aβ plaques and finding new compounds which decrease the AChE-Aβ interaction may be a putative therapeutic agent to fight the disease.

Multiple cascade effects of oxidative stress on astroglia

Many neurodegenerative diseases share common underlying features, most prominent of which are dysregulation of calcium homeostasis and reactive astrogliosis, ultimately triggered by oxidative stress. Interestingly, an additional feature of the early response to stress conditions is the upregulation and release of acetylcholinesterase (AChE). This study therefore investigates the link between oxidative stress, calcium influx, gene expression, protein synthesis, and AChE release. We report that, in astroglia and in an immortalized cell line, GH4-halpha7, acute oxidative stress causes influx of extracellular calcium through L-type voltage-gated calcium channels (L-VGCC), followed by increased release of AChE into the extracellular medium. Moreover, rapid and sustained changes in mRNA expression of AChE, L-VGCC, and melastatin-like transient receptor potential 2 (TRPM2) accompany profound suppression of global protein synthesis. Application of L-VGCC blockers selectively reduces stress-induced calcium influx and AChE release, mitigates changes in gene expression, and facilitates recovery from protein synthesis suppression. Although glia exhibit greater sensitivity in their responses, the results are comparable in astroglia and GH4-halpha7 cells, and suggest a generalized and integrated cellular response to stress conditions that characterizes changes observed in neurodegeneration.

Gene expression signature of parathion-transformed human breast epithelial cells

Environmental substances seem to be involved in the etiology of breast cancers. Many studies have found an association between human cancer and exposure to agricultural pesticides such as the organophosphorous pesticides. Parathion is a cholinesterase inhibitor that induces the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. The primary target of action in insects is the nervous system whereby pesticides inhibit the release of the enzyme acetylcholinesterase at the synaptic junction. Atropine is a parasympatholytic alkaloid used as an antidote to acetylcholinesterase inhibitors. The aim of this study was to determine the effect of parathion and atropine on cell transformation of human breast epithelial cells in vitro. These studies showed that parathion alone was able to induce malignant transformation of an immortalized human breast epithelial cell line, MCF-10F as indicated by increased cell proliferation, anchorage independency and invasive capabilities. There was also an increase in c-kit, Trio, Rho-A, Rac-3, EGFR, Notch-4, Dvl-2, Ezrin, beta catenin and mutant p53 protein expression in the parathion-treated cells. However, atropine significantly inhibited this increase. In a human cell cycle array of 96 genes, 13 of them were altered by parathion treatment. Among the genes affected were the cyclins, such as cyclin D3, the cyclin-dependent kinases (CDKs) such as CDK41 and the minichromosome maintenance deficient (MCM) MCM2 and MCM3. It is suggested that parathion influences human breast epithelial cell transformation and is an initiator factor in the transformation process in breast cancer.

A peptide derived from the C-terminal region of acetylcholinesterase modulates extracellular concentrations of acetylcholinesterase in the rat substantia nigra

It is well established that acetylcholinesterase (AChE) has ‘non-classical’ functions independent of cholinergic transmission. A region of AChE distinct from the catalytic site may be responsible for these actions via a 14-residue peptide located between residues 586–599 at the C-terminus of human AChE. This AChE-peptide possesses a high amino acid sequence homology with a region of amyloid precursor protein and shares many biophysical and physiological characteristics. In this study, the effect of AChE-peptide (AEFHRWSSYMVHWK) on the extracellular levels of endogenous AChE was examined in rat substantia nigra in vitro. A chemiluminescent assay was used to continuously measure the soluble AChE concentration from tissue punches of the substantia nigra. Application of NMDA evoked an increase in extracellular AChE levels consistent with previous results obtained from in vivo models. AChE-peptide, when applied alone, had no effect on AChE release: however, when co-applied with NMDA, AChE-peptide reduced the effectiveness of NMDA to evoke release of AChE. These results indicate, in a region of the brain central to the aetiology of Parkinson's disease, that an AChE-peptide fragment derived from AChE displays a bioactivity that could involve regulation of Ca 2+ availability and hence the release of AChE.

Diethylenetriaminopentaacetic acid is unsuitable for long-term preservation of RBCs.

The addition of an appropriate metal chelator, such as diethylenetriaminopentaacetic acid (DTPA) to stored blood has been shown to be effective in a short-term (0-12 days) prevention of lipid peroxidation of stored RBCS: However, its long-term effectiveness has not been carefully evaluated. Blood was preserved in simulated blood bank conditions with or without the addition of DTPA for 4 weeks. Aliquots of stored blood were taken weekly from the storage bag and the deformability profile was determined using a custom-built laser viscodiffractometer. Malondialdehyde (MDA), an index of lipid peroxidation, and the extent of vesiculation of the stored blood were quantified concurrently. It was found that MDA values for DTPA-supplemented blood at the end of a 28-day storage period were significantly elevated compared with the DTPA-free counterpart (23.50 +/- 3.2 vs. 16.10 +/- 2.5 microM; p<0.05). In addition, DTPA-supplemented blood was more susceptible to vesiculation than its DTPA-free counterpart (31.26 +/- 4.1 vs. 10.26 +/- 1.5% of acetyl cholinesterase release, p<0.001). These data are also in accordance with the finding of the deformability profile result, indicating that DTPA-supplemented blood exhibits not only a decrease in deformability index, but also a tendency to shift the profile to a lower osmolality compared with that of controls (a dehydration phenomenon). Long-term (0-28 days) preservation of human RBCs with DTPA caused a gradual increase in MDA production, a progressive enhancement of the severity of vesiculation, and an alteration in the deformability profile. Free-radical-mediated oxidative damage is likely to be the culprit for this observed phenomenon. In addition, the direct effect of DTPA on RBC structural integrity must be considered.

Induction of Acetylcholinesterase Release from Erythrocytes in the Presence of Liposomes

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.

Rat Locomotion and Release of Acetylcholinesterase

In the substantia nigra acetylcholinesterase is released from the dopamine cells of the pars compacta independent of cholinergic transmission. In this study the effects of local and systemic amphetamine treatment were compared on acetylcholinesterase release in the rat substantia nigra in relation to concomitant behavior. Acetylcholinesterase release, measured “on-line” with a sensitive chemiluminescent system, was enhanced by amphetamine stimulation administered locally and could not be dissociated from simultaneous amphetamine-induced circling behavior. On the other hand, amphetamine administered systemically resulted in a general increase in locomotor behavior followed by a subsequent increase in acetylcholinesterase release. The alternative scenario of an initial rise in acetylcholinesterase release, subsequently followed by enhanced movement, was never seen. Hence, movement can enhance release of acetylcholinesterase from the substantia nigra, whereas “upstream” local nigral events can affect acetylcholinesterase release and movement simultaneously.

Motor neurone acetylcholinesterase release precedes neurotoxicity caused by systemic administration of excitatory amino acids and strychnine.

We have proposed that neuronal overactivation by either stimulation of excitatory receptors or hypofunction of inhibitory circuits is a cause of excessive acetylcholinesterase (AChE) release, which, in turn, can contribute to ALS/MND pathogenesis. We investigated histochemical and histopathological changes in cell populations of the mouse spinal ventral horn upon in vivo stimulation of glutamate receptors with L-aspartate (ASP, 10-50 mg/kg, intraperitoneal: i.p.), or blockade of glycine receptors with strychnine (STRY, 2 mg/kg, i.p.). ASP in P4-P13 (postnatal age in days) but not in older mice, and STRY irrespective of age, provoked rapid, striking depletions of motor neurone AChE, and appearance of AChE activity in astrocytes. This was followed by recovery of the enzyme in most motor neurones, astrocyte activation and statistically significant changes in: brain macrophage infiltration, loss of interneurones and motor neurones and neuronophagic images including rosettes of glial cells surrounding a central 'ghost-like' motor neurone. Although AChE release preceded the neuropathology found, it is not known if its uptake is a cause of glial activation. However, it has been shown that the enzyme potentiates non-N-metyl-D-aspartate receptors identical to those that mediate astrocyte activation. AChE activity produces protons and choline, possible microglial activators. These are putative routes towards long-lasting neuropathology.