Sternfeld MeiraHebrew University of Jerusalem, Dept Biological Chemistry, Institute of Life Sciences, 91904 Jerusalem IsraelPhone : Fax : Send E-Mail to Sternfeld Meira
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 AChE-R 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.
Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).
Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The "read-through" variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the "synaptic" AChE variant, AChE-S. Several observations demonstrate that excess AChE-R attenuates, whereas AChE-S intensifies, neurodeterioration. In the somatosensory cortex, AChE-S transgenics, but not AChE-R or control FVB/N mice, displayed a high density of curled neuronal processes indicative of hyperexcitation. In the hippocampus, AChE-S and control mice, but not AChE-R transgenics, presented progressive accumulation of clustered, heat shock protein 70-immunopositive neuronal fragments and displayed a high incidence of reactive astrocytes. Our findings suggest that AChE-R serves as a modulator that may play a role in preventing the shift from transient, acute stress to progressive neurological disease.
Title: Structural roles of acetylcholinesterase variants in biology and pathology Grisaru D, Sternfeld M, Eldor A, Glick D, Soreq H Ref: European Journal of Biochemistry, 264:672, 1999 : PubMed
Apart from its catalytic function in hydrolyzing acetylcholine, acetylcholinesterase (AChE) affects cell proliferation, differentiation and responses to various insults, including stress. These responses are at least in part specific to the three C-terminal variants of AChE which are produced by alternative splicing of the single ACHE gene. 'Synaptic' AChE-S constitutes the principal multimeric enzyme in brain and muscle; soluble, monomeric 'readthrough' AChE-R appears in embryonic and tumor cells and is induced under psychological, chemical and physical stress; and glypiated dimers of erythrocytic AChE-E associate with red blood cell membranes. We postulate that the homology of AChE to the cell adhesion proteins, gliotactin, glutactin and the neurexins, which have more established functions in nervous system development, is the basis of its morphogenic functions. Competition between AChE variants and their homologs on interactions with the corresponding protein partners would inevitably modify cellular signaling. This can explain why AChE-S exerts process extension from cultured amphibian, avian and mammalian glia and neurons in a manner that is C-terminus-dependent, refractory to several active site inhibitors and, in certain cases, redundant to the function of AChE-like proteins. Structural functions of AChE variants can explain their proliferative and developmental roles in blood, bone, retinal and neuronal cells. Moreover, the association of AChE excess with amyloid plaques in the degenerating human brain and with progressive cognitive and neuromotor deficiencies observed in AChE-transgenic animal models most likely reflects the combined contributions of catalytic and structural roles.
Title: Transgenic mammary gland expression of readthrough human AChE: A model system for cholinesterase regulation in mammalian body fluids Salmon AY, Sternfeld M, Ginzberg D, Patrick J, Soreq H Ref: Journal de Physiologie (Paris), 92:489 , 1998 : PubMed
Title: Human acetylcholinesterase transgenic mice show elevated muscle expression and brain specific silencing Sternfeld M, Patrick JD, Soreq H Ref: Journal de Physiologie (Paris), 92:499, 1998 : PubMed
Title: Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini Sternfeld M, Ming G, Song H, Sela K, Timberg R, Poo MM, Soreq H Ref: Journal of Neuroscience, 18:1240, 1998 : PubMed
Accumulated indirect evidence suggests nerve growth-promoting activities for acetylcholinesterase (AChE). To determine unequivocally whether such activities exist, whether they are related to the capacities of this enzyme to hydrolyze acetylcholine and enhance synapse development, and whether they are associated with alternative splicing variants of AChEmRNA, we used four recombinant human AChEDNA vectors. When Xenopus laevis embryos were injected with a vector expressing the synapse-characteristic human AChE-E6, which contains the exon 6-encoded C terminus, cultured spinal neurons expressing this enzyme grew threefold faster than co-cultured control neurons. Similar enhancement occurred in neurons expressing an insertion-inactivated human AChE-E6-IN protein, containing the same C terminus, and displaying indistinguishable immunochemical and electrophoretic migration properties from AChE-E6, but incapable of hydrolyzing acetylcholine. In contrast, the nonsynaptic secretory human AChE-I4, which contains the pseudointron 4-derived C terminus, did not affect neurite growth. Moreover, no growth promotion occurred in neurons expressing the catalytically active C-terminally truncated human AChE-E4, demonstrating a dominant role for the E6-derived C terminus in neurite extension. Also, AChE-E6 was the only active enzyme variant to be associated with Xenopus membranes. However, postsynaptic length measurements demonstrated that both AChE-E6 and AChE-E4 enhanced the development of neuromuscular junctions in vivo, unlike the catalytically inert AChE-E6-IN and the nonsynaptic AChE-I4. These findings demonstrate an evolutionarily conserved synaptogenic activity for AChE that depends on its hydrolytic capacity but not on its membrane association. Moreover, this synaptogenic effect differs from the growth-promoting activity of AChE, which is unrelated to its hydrolytic capacity yet depends on its exon 6-mediated membrane association.
Title: Position effect variegations and brain-specific silencing in transgenic mice overexpressing human acetylcholinesterase variants Sternfeld M, Patrick JD, Soreq H Ref: Journal de Physiologie (Paris), 92:249, 1998 : PubMed
Position effect variegations as well as brain-specific silencing were observed in novel transgenic mouse pedigrees expressing human acetylcholinesterase (AChE) variants. Muscle AChE activities varied between 1.6- and 350-fold of control in these lines, one carrying insertion-inactivated InE6-AChE and two with 'readthrough' I4/E5 AChE, all under control of the ubiquitous CMV promoter. In contrast, brain AChE levels remained within a range of 1.5-fold over control, suggesting an upper limit of brain AChE which is compatible with life.
1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption 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 alpha-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 alpha-solanine. However, BuChE in the serum of the 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 of the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.
Title: Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells Karpel R, Sternfeld M, Ginzberg D, Guhl E, Graessmann A, Soreq H Ref: Journal of Neurochemistry, 66:114, 1996 : PubMed
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 14/E5 region induced the appearance of small, round cells in a dominant manner. This was associated with expression of 14/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.
Butyrylcholinesterase [BCHE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BCHE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BCHE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BCHE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BCHE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.
Tissue-specific heterogeneity among mammalian acetylcholinesterases (AChE) has been associated with 3' alternative splicing of the primary AChE gene transcript. We have previously demonstrated that human AChE DNA encoding the brain and muscle AChE form and bearing the 3' exon E6 (ACHE-E6) induces accumulation of catalytically active AChE in myotomes and neuromuscular junctions (NMJs) of 2- and 3-day-old Xenopus embryos. Here, we explore the possibility that the 3'-terminal exons of two alternative human AChE cDNA constructs include evolutionarily conserved tissue-recognizable elements. To this end, DNAs encoding alternative human AChE mRNAs were microinjected into cleaving embryos of Xenopus laevis. In contrast to the myotomal expression demonstrated by ACHE-E6, DNA carrying intron 14 and alternative exon E5 (ACHE-I4/E5) promoted punctuated staining of epidermal cells and secretion of AChE into the external medium. Moreover, ACHE-E6-injected embryos displayed enhanced NMJ development, whereas ACHE-I4/E5-derived enzyme was conspicuously absent from muscles and NMJs and its expression in embryos had no apparent effect on NMJ development. In addition, cell-associated AChE from embryos injected with ACHE-I4/E5 DNA was biochemically distinct from that encoded by the muscle-expressible ACHE-E6, displaying higher electrophoretic mobility and greater solubility in low-salt buffer. These findings suggest that alternative 3'-terminal exons dictate tissue-specific accumulation and a particular biological role(s) of AChE, associate the 3' exon E6 with NMJ development, and indicate the existence of a putative secretory AChE form derived from the alternative I4/E5 AChE mRNA.
Title: Alternative Exon 6 Directs Synaptic Localization of Recombinant Human Acetylcholinesterase in Neuromuscular Junctions of Xenopus laevis Embryos Sternfeld M, Seidman S, Ben Aziz-Aloya R, Shapira M, Timberg R, Kaufer D, Soreq H Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:45, 1995 : PubMed
To study the molecular mechanisms underlying the intensive expression of acetylcholinesterase (AChE) in different tumor types, we characterized levels and composition of its messenger RNA (mRNA) sequences in heterologous tumor cell lines, primary tumor biopsies, and normal fetal and adult tissues and determined their exon-intron origin within the corresponding ACHE gene. Reverse transcription followed by polymerase chain reaction (RT-PCR) revealed three alternatively spliced ACHE mRNAs in NT2/D1 teratocarcinoma, NCI-N-592 small cell lung carcinoma, TE671 medulloblastoma, K-562 erythroleukemia, and 293 transformed embryonal kidney cells. The three ACHE mRNAs include the principal species expressed in brain and muscle and two additional transcripts containing insertions of 751 or 829 residues downstream from the exon 4 domain. The inserted region, which represents an intron in brain and muscle, is expressed in the tumor cell lines either as a "readthrough" form or with 78 residues deleted from its 5' end. A major band of 2.5 kb was labeled with ACHE cDNA in poly(A)+ RNA blots from medulloblastoma cells or brain tissue, whereas a PCR-amplified probe from the inserted domain labeled a 3.4-kb band but not the 2.5-kb band in poly(A)+ RNA from small cell lung carcinoma. The ACHE mRNAs including the alternative insertions were found only in cell lines with levels of the principal ACHE mRNA species equal to or higher than those in brain (1-10 molecules/cell), determined by following the kinetics of mRNA PCR amplification. Genomic DNA sequencing revealed that the inserted domains in the ACHE mRNAs expressed in the tumor cell lines encode C-terminal peptides of 40 and 14 residues. These include a free cysteine, terminate with the consensus HG element, and continue by a 29-residue-long C-terminal hydrophobic cleavable peptide, properties characteristic of precursors to phosphoinositide (PI)-linked proteins. In extension of the reported expression of PI-linked AChE in hemopoietic cells including K-562, our findings demonstrate the existence of ACHE mRNAs with the potential to encode one hydrophilic and two PI-linked forms of AChE in tumor cells from both hemopoietic and nonhemopoietic origins.
Title: Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins Shapira M, Seidman S, Sternfeld M, Timberg R, Kaufer D, Patrick J, Soreq H Ref: Proceedings of the National Academy of Sciences of the United States of America, 91:9072, 1994 : PubMed
To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximately 2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 microns2 in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with average postsynaptic lengths up to 1.8-fold greater than controls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were > 2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.
Title: Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos Ben-Aziz-Aloya R, Seidman S, Timberg R, Sternfeld M, Zakut H, Soreq H Ref: Proceedings of the National Academy of Sciences of the United States of America, 90:2471, 1993 : PubMed
We have employed Xenopus embryos to express human acetylcholinesterase (AcChoEase; EC 3.1.1.7) in developing synapses. Transcription of AcChoEase mRNA was driven by a 2.2-kb sequence upstream from the initiator AUG in the ACHE gene encoding AcChoEase, with multiple potential sites for binding universal and tissue-specific transcription factors. These included clustered MyoD elements, E-box, SP1, EGR1, AP-2, and the development-related GAGA motif. A DNA construct composed of this sequence linked to a 2.1-kb sequence encoding human AcChoEase was designated human AcChoEase promoter-reporter (HpACHE). HpACHE but none of its several 5'-truncated derivatives was transcriptionally active in developing Xenopus embryos. Furthermore, PCR analysis using chimeric PCR primers revealed usage of the same 1.5-kb intron and 74-bp exon within the HpACHE sequence in microinjected embryos and various human tissues. Cytochemical staining revealed conspicuous accumulation of overexpressed AcChoEase in neuromuscular junctions and within muscle fibers of apparently normal 2-day Xenopus embryos injected with HpACHE. The same reporter driven by the cytomegalovirus promoter was similarly efficient in directing the heterologous human enzyme toward neuromuscular junctions, attributing the evolutionary conservation of AcChoEase targeting to the coding sequence. Our findings demonstrate that a short DNA sequence is sufficient to promote the exogenous transcription and faithful splicing of human AcChoEase mRNA in developing Xenopus embryos and foreshadow their use for integrative studies of cholinergic signaling and synapse formation.
Title: Promoter elements and alternative splicing in the human ACHE gene Ben-Aziz-Aloya R, Sternfeld M, Soreq H Ref: Progress in Brain Research, 98:147, 1993 : PubMed
