Meira Sternfeld

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.

ARP, a peptide derived from the stress-associated acetylcholinesterase variant has hematopoietic growth promoting activities

BackgroundPsychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated “readthrough” acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses.Materials and MethodsWe studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD341 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors.ResultsWe identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD341 hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages.ConclusionsOur findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress‐related infertility are not yet known. Overexpression of the “readthrough” variant of acetylcholinesterase (AChE‐R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE‐R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age‐matched control mice. Transgenic mice overexpressing AChE‐R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age‐matched nontransgenic controls. AChE‐R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE‐R transgenic mice. Head‐localized AChE‐R was characteristic of human sperm from fertile donors. In contrast, sperm head AChER staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE‐R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress‐related infertility.

Normal and Atypical Butyrylcholinesterases in Placental Development, Function, and Malfunction

Abstract1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous abortion and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development.2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta.3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with α-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction.4. Atypical BuChE of serum or recombinant origin presented >10-fold weaker affinities than normal BuChE for cocaine and α-solanine. However, BuChE in the serum of a heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta.5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls or the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Molecular Dissection of Protein Domains Directing the Tissue Targeting of Acetylcholinesterase Isoforms

Acetylcholinesterase (AChE) accumulates in neuromuscular junctions (NMJs) where it terminates synaptic transmission by hydrolyzing the neurotransmitter acetylcholine (ACh) (Salpeter, 1967). In addition, AChE is expressed in non-cholinergic tissues such as the hematopoietic system (Paulus et al., 1981), germ cells (Gundersen and Miledi, 1983), embryonic tissues at developmental stages before the onset of cholinergic transmission (Layer, 1991) and malignant tumors (Karpel et al., 1994). The role of AChE in these non-cholinergic tissues is unknown and is sometimes speculated to be non-catalytical. ACHE, the human gene encoding AChE, is located on the long arm of chromosome 7 (7q22) (Getman et al., 1992; Ehrlich et al, 1992), and spans a region of 7 Kb. Three 3′ — alternative splicing options yield 3 mature AChE mRNAs whose catalytically active protein products differ at their C-termini (Ben Aziz-Aloya et al., 1993; Karpel et al., 1994).

A peptide cleaved from the stress associated variant form of acetylcholinesterase modulates hematopoiesis

Abstract Hematopoietic stress responses involve increases in white blood cells (WBC) and platelet counts, implying the existence of stress responsive factors that modulate hematopoiesis. Acetylcholinesterase AChE, which terminates neurotransmission in synapses, is also expressed in several hematopoietic lineages and has been postulated to play a role in hematopoiesis. In support of this notion we report that the extended promoter region of the ACHE gene contains both hematopoietic transcription factor binding sites and stress associated glucocorticoid response element (GRE) half-palindromic sites. An intriguing stress-response candidate is the readthrough isoform AChE-R which is produced by alternative splicing of the ACHE gene. This soluble monomeric variant is expressed in embryonic and tumor cells, in mammalian hematopoietic cells and is induced in the brain under psychological, physical and chemical stress (Kaufer et al, Nature 393:373, 1998, Grisaru et al. FEBS 264:672, 1999). We found that following forces swim stress, a c-terminal peptide (ARP) is cleaved from ACHE-R which accumulates in mouse blood. Injection of synthetic ARP in mice augmented stress-associated blood cell expansion, while antisense suppression of AChE-R mRNA blocked stress-induced accumulation of ARP in mouse bone marrow (BM) and suppressed hematopoietic stress responses. In vitro , synthetic ARP (2 nM) induced survival and stimulated expansion of human CD34 + cells, and early myeloid and megakaryocyte progenitors. Moreover, transgenic mice overexpressing AChE-R presented elevations in BM progenitors and WBC and platelet counts. Our findings present ARP, the C-terminal peptide of AChE-R, as a new hematopoietic growth factor which may promote myeloid and megakaryocytic expansion characteristic of stress.

Catalytic and Non-Catalytic Activities of Acetylcholinesterase Implied from Transgenic ACHE Expression in Vertebrates

Transgenic overexpression of human acetylcholinesterase (AChE) was employed as an experimental tool for creating a subtle cholinergic imbalance in vertebrates. In Xenopus laevis tadpoles, transgenic AChE accelerated the development of neuromuscular junctions. In stably transgenic mice, overexpression of AChE was associated with deterioration in both cognitive and neuromotor functions, suggesting requirement for balanced cholinergic transmission for stable maintenance of these functions. However, transfections of cultured glioma cells demonstrated a morphogenic activity for AChE which was apparently non-catalytic. This suggested that putatively distinct feedback mechanisms are activated in response to the catalytic and non-catalytic activities of excess AChE, which causes the observed symptoms.

Central and Peripheral Consequences of Cholinergic Imbalance in Alzheimer’s Disease

Cholinergic neurotransmission in the central and the peripheral nervous system is a primary feature common to all vertebrates (Soreq and Zakut, 1993). Rapid, fine tuning sustains well balanced ratios between key cholinergic constituents during cognitive processes such as learning and memory. To assure such higher brain functions, the levels of acetylcholine (ACh) should be tightly regulated. This is achieved by maintenance of a dynamic equilibrium between the levels of the synthesizing enzyme choline acetyl transferase (ChAT), the hydrolyzing enzyme acetylcholinesterase (AChE; Schwarz et al., 1995) and the high affinity choline transporter responsible for the re-uptake of choline (Slotkin et al., 1994). Changes in this equilibrium occur in physiopathological syndromes such as Alzheimer’s disease (AD) (Beeri et al., 1995, Andres et al., 1996). Cholinergic imbalance is associated with characteristic modifications in the electrophysiological properties of certain regional circuits and consequent disruption of central transmission systems. Therefore, perturbed cholinergic communication may cause long-term central responses affecting cognitive, autonomous and neuromotor performance. To delineate the molecular mechanisms leading from cholinergic imbalance to dysfunction, we combined transgenic and gene expression tests with electrophysiological and molecular biology analyses in in vivo and in vitro (brain slice) systems from various species.

Alternative Exon 6 Directs Synaptic Localization of Recombinant Human Acetylcholinesterase in Neuromuscular Junctions of Xenopus laevis Embryos

We have expressed and characterized catalytically active recombinant human acetylcholinesterase (rHAChE) produced in microinjected oocytes of Xenopus laevis. However, the highly specialized, single-cell nature of the oocyte limits its usefulness in addressing questions regarding tissue-specific processing and the biological roles of cloned nervous system proteins. Therefore, to study the role of 3’ alternative splicing in regulating tissue-specific expression of the human ACHE gene encoding AChE, we established an in vivo model in transiently transgenic Xenopus embryos. Following injection into in vitro fertilized Xenopus eggs, whole-mount cytochemistry and electron microscopy revealed that ACHE DNA bearing the alternative 3’ exon E6 (ACHE-E6) induced prominent overexpression of catalytically active AChE in myotomes of 2- and 3-day-old embryos, including neuromuscular junctions (NMJs). NMJs from ACHE-E6-injected embryos displayed, on average, 4-fold greater AChE-stained areas (SA) and 80% increased post-synaptic lengths (PSL) compared to age-matched uninjected controls. Perhaps more significantly, ACHE-E6 overexpression stimulated the appearance of a class of large NMJs (PSL>4 mm) rarely observed in control embryos, apparently at the expense of small (PSL<3 mm) NMJs. Homogenates prepared from these embryos demonstrated increased binding of biotinylated a-bungarotoxin, indicating enhanced expression of the endogenous Xenopus acetylcholine receptor and suggesting coordinated regulation of cholinergic proteins in the developing NMJ. When exon E6 was replaced by ACHE DNA encoding the pseudo-intron I4 and 3’ alternative exon E5, overexpressed rHAChE accumulated in epidermal cells, but not in muscle or NMJs. These findings, therefore, attribute an evolutionarily conserved NMJ-accumulating role for exon E6 and provide in vivo evidence for tissue-specific management of alternative AChEs.