Efrat Lev-Lehman

Transgenic expression of human acetylcholinesterase induces progressive cognitive deterioration in mice

BACKGROUND Cognitive deterioration is a characteristic symptom of Alzheimers disease. This deterioration is notably associated with structural changes and subsequent cell death which occur, primarily, in acetylcholine-producing neurons, progressively damaging cholinergic neurotransmission. We have reported previously that excess acetylcholinesterase (AChE) alters structural features of neuromuscular junctions in transgenic Xenopus tadpoles. However, the potential of cholinergic imbalance to induce progressive decline of memory and learning in mammals has not been explored. RESULTS To approach the molecular mechanisms underlying the progressive memory deficiencies associated with impaired cholinergic neurotransmission, we created transgenic mice that express human AChE in brain neurons. With enzyme levels up to two-fold higher than in control mice, transgenic mice displayed an age-independent resistance to the hypothermic effects of the AChE inhibitor, paraoxon. In addition to this improved scavenging capacity for anti-AChEs, however, these transgenic mice also resisted muscarinic, nicotinic and serotonergic agonists, indicating that secondary pharmacological changes had occurred. The transgenic mice also developed progressive learning and memory impairments, although their locomotor activities and open-field behaviour remained similar to those of matched control mice. By six months of age, transgenic mice lost their ability to respond to training in a spatial learning water maze test, whereas they performed normally in this test at the age of four weeks. This animal model is therefore suitable for investigating the transcriptional changes associated with cognitive deterioration and for testing drugs that may attenuate progressive damage. CONCLUSION We conclude that upsetting cholinergic balance may by itself cause progressive memory decline in mammals, suggesting that congenital and/or acquired changes in this vulnerable balance may contribute to the physiopathology of Alzheimers disease.

Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3' alternative splicing variant of acetylcholinesterase.

ABSTRACT The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)2 vitamin D3 and 17β-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3′ terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors

Summary1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls.2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range.3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment.4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death.5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis.6.In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown byin situ hybridization.7.Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment.8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzymes activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.

Cholinotoxic effects on acetylcholinesterase gene expression are associated with brain-region specific alterations in G,C-rich transcripts

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

54 Neuromuscular deterioration in ache-transgenic mice

The long-term contribution of balanced cholincrgic neurotransmission toward neuromuscular properties was studied in transgcnic mice b) expressing human acctylcholincsterasc (hAChE) in motoneurons, but not muscle. Spinal cord Cholinergic axodcndritic synapses in transgcnic mice wverc morphologically normal despite 7-fold higher AChE activity staining as compared with control synapses In contrast, although muscle extracts included only ca. 6%) of hAChE from motoneuron origin, transgcnic neuromuscular junctions were 60% larger than control ones and displayed either exaggerated or degenerated post-synaptic folds Neuromuscular impairment uas evident in grip tests at the age of 1 weeks, worsened with age and was accompanied by progressive amyotrophy and abnormal electromyogmphic potentials, rcflccting cnlargcd motor units and junctional dysfunction. The vulnerability of vertebrate neuromuscular .junctions to alterations in cholinergic ncurotransmission, highlights the morphogenic role of AChE Human AChE-expressing mice can hence be used for dissecting tllC molecular mechanisms underlying neuromuscular proprrties. FUNCTIONAL STUDIES OF THE SYNAPTIC VESICLE PROTEINS

Transient Alterations in the in Vivo Levels of Cholinesterase mRNAS Suggest Differential Adjustment to Cholinotoxic Stimuli

Numerous diseases of the central nervous system (CNS) are associated with cholinergic deficits, Alzheimer’s disease being a notable example of such neurodegenerative disorders (Wurtman, 1992). To dissect the molecular mechanisms involved in the impairment of cholinergic neurotransmission in this and other CNS diseases, experimental approaches should be pursued which combine in vivo model systems with sensitive, multileveled detection methods.

Structure-Function Relationships, In Vivo Mutability and Gene Amplification in Human Cholinesterases, Targets for Organophosphorous Poisons

The human Cholinesterase genes and their protein products have been the focus of intensive research for many years (for comprehensive reviews see Whittaker, 1986; 1Rakonczay and Brimijoin, 1988; and Soreq and Zakut, 1990) because of the physiological function attributed to these enzymes, which are both capable of hydrolyzing the neurotransmitter acetylcholine. Genetic linkage evidence indicated that two distinct genes, designated ACHE and CHE, encode the two principal forms of cholinesterases, acetylcholinesterase (acetylcholine acetyl hydrolase, AChE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, BuChE, EC 3.1.1.8) which differ in their substrate specificities and sensitivities to selective inhibitors. The toxic effects of organophosphorous (OP) poisons, such as common insecticides or nerve gases, are generally attributed to their specific inhibition of cholinesterases, interfering with cholinergic neurotransmission. OP inhibition of cholinesterases occurs through a covalent interaction of the OP compounds with a serine residue in the active esteratic site (Koelle, 1972). However, detailed structure-function relationships in this family of enzymes have been hampered by the difficulties in purifying mammalian cholinesterases.