Dalia Ginzberg

Overexpression of Alternative Human Acetylcholinesterase Forms Modulates Process Extensions in Cultured Glioma Cells

Abstract: In addition to its well‐known synaptic function, acetylcholinesterase was recently shown to stimulate neurite outgrowth from cultured chick neurons in a manner unrelated to its catalytic activity. It remained unclear, however, whether each of the variant acetylcholinesterase enzyme forms can promote such process extension and whether this effect of acetylcholinesterase was limited to neurite outgrowth. Using DNA microinjections and stable transfections of cultured glioma cells, we explored the possibility that specific acetylcholinesterase isoforms affect cellular development and morphology of CNS astrocytes. Cells microinjected with human ACHEDNA constructs that differ in their exon‐intron composition displayed rapid yet stable induction of cell body enlargement and process extensions. Cells transfected with ACHEDNA carrying the neuronal‐characteristic 3′‐E6 domain also displayed stable process extensions. However, stable transfections with ACHEDNAs including the 3′‐alternative I4/E5 region induced the appearance of small, round cells in a dominant manner. This was associated with expression of I4/E5‐ACHEmRNA transcripts and the production of soluble acetylcholinesterase monomers that were catalytically indistinguishable from the 3′‐E6 enzyme but displayed higher electrophoretic mobility than that of the 3′‐E6 form. Thus, variable expression levels and alternative splicing modes of the ACHE gene correlated in these experiments with glial development in a manner that was apparently unrelated to catalysis.

Endotoxin-induced changes in human working and declarative memory associate with cleavage of plasma “readthrough” acetylcholinesterase

Endotoxin stimulation of the immune system produces marked alterations in memory functioning. However, molecular links between this cognitive response and infection-responding neurotransmission pathways are still unknown. The cytokine and memory responses of volunteers injected with 0.8 ng/kg Salmonella endotoxin were compared with changes in plasma levels and integrity of the stress-induced acetylcholinesterase variant, AChE-R. Vascular endothelial cells were found to express AChE-R messenger RNA and protein both in healthy and inflamed human tissues. Plasma AChE activity was reduced after endotoxin treatment, but not placebo treatment, parallel to the decline in cortisol after the endotoxin-induced peak and inversely to the accumulation of a C-terminal immunopositive AChE-R peptide of 36 amino acid residues. AChE-R cleavage coincided with significant endotoxin-induced improvement in working memory and impairment in declarative memory. By 3 h posttreatment, working memory improvement was negatively correlated with AChE-R cleavage, which showed association to proinflammatory cytokine levels. By 9 h posttreatment, declarative memory impairment was negatively correlated with AChE-R cleavage and positively correlated with the suppressed AChE activity. Endotoxin-induced peripheral cholinergic stress responses are hence associated with greater impairment in declarative memory and lower improvement in working memory, pointing at AChE-R as a surrogate marker of psychoneuroimmunological stress.

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzymes affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

Summary1.Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities.2.Homologous cosmid recombination with a 190-base pair 5′ fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3′ end of BuChEcDNA from nervous system tumors and which has been mapped byin situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns.3.The chromosomal origin of BuChE-coding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringentin situ hybridization protocol, including washes at high temperature and low salt.4.These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family of highly similar proteins encoded by distinct, nonhomologous genes.

Metabolic aspects of LPP cyanophage replication in the cyanobacterium Plectonema boryanum

Cyanophage LPP1G is reproduced at the same yield in heterotrophic conditions (dark, glucose) as in photoautotrophic conditions; aerobiosis is required for dark cyanophage replication. Exogenous glucose is not required for the cyanophage replication in the dark in heterotrophically grown cells. In photoautotrophically grown cells, the maximum burst size in dark and glucose is delayed for a period corresponding to glucose uptake induction. Cyanophage LPP2SPI replication occurs in conditions where only Photosystem I operates. Of photosynthesis parameters tested, only CO2 photoassimilation is affected during cyanophage LPP1G infection under photoautotrophic conditions.

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.

Testicular Gene Amplification and Impaired BCHE Transcription Induced in Transgenic Mice by the Human BCHE Coding Sequence

Multiple findings implicate acetylcholine with sperm functioning 1,2 and acetyl-and butyrylcholinesterase activities (ACHE, BCHE) were observed in mammalian sperm cells and during oocyte development 1–3. In vivo amplification of the human BCHE gene was first found in a father and son exposed to cholinesterase inhibitors 4, but it remained unclear whether the amplified DNA was transmitted as such from father to son or whether the amplification phenomenon re-occurred in germ cells, particularly during male meiosis or sperm differentiation.

Antisense Oligodeoxynucleotide Dependent Suppression of Acetylcholinesterase Expression Reduces Process Extension from Primary Mammalian Neurons

The only currently approved drugs for Alzheimer’s disease (AD) are potent blockers of acetylcholinesterase (AChE) activity (Knapp et al., 1994). However, several lines of evidence suggest novel, non-catalytic morphogenic properties of AChE in process extension (Small et al, 1995; Layer and Willbold, 1995; Jones et al., 1995; Darboux et al., 1996; Sternfeld et al., 1997) and amyloid fibril formation (Inestrosa et al, 1996). This calls for the development of alternative approaches in which both AChE protein synthesis and enzymatic activity would be suppressed, such as the “antisense”.technology (Grifman et al., 1997). To this end, we have designed seven synthetic 3′-phosphorothioatedoligonucleotides (AS-ODNs) targeted towards AChEmRNA and tested their AChE suppression efficacies on the rat neuroendocrine pheochromocytoma cell line, PC 12. Two of these AS-ODNs suppressed the catalytic activity of AChE in nerve growth factor (NGF) -treated PC 12 cells by 25–35%, significantly more than the parallel suppression by control ODNs (Grifman and Soreq, 1997). To study the involvement of AChE in neurite outgrowth and differentiation of primary neurons, we added these two AS-ODNs to primary neuronal cultures from embryonic (El4) mouse whole brain.

The Cholinotoxin AF64A Differentially Attenuates in Vitro Transcription of the Human Cholinesterase Genes

Numerous reports in the literature have now demonstrated that ethylcholine aziridinium (AF64A) exerts selective cholinotoxicity, in vivo, in a number of animal species.1,2 These effects are dose-and time-dependent and reversible, when low concentrations of AF64A (e.g. ≤ 2 nmol/lateral ventricle in the rat) are used.3 The dose range for cholinoselectivity of AF64A needs to be established accurately with each specific application, since there is an upper dose limit at which AF64A begins to exert nonspecific degenerative effects.1