Shlomo Seidman

Acute stress facilitates long-lasting changes in cholinergic gene expression

Acute traumatic stress may lead to post-traumatic stress disorder (PTSD), which is characterized by delayed neuropsychiatric symptoms including depression, irritability, and impaired cognitive performance. Curiously, inhibitors of the acetylcholine-hydrolysing enzyme acetylcholinesterase may induce psychopathologies that are reminiscent of PTSD,. It is unknown how a single stressful event mediates long-term neuronal plasticity. Moreover, no mechanism has been proposed to explain the convergent neuropsychological outcomes of stress and of acetylcholinesterase inhibition. However, acute stress elicits a transient increase in the amounts released of the neurotransmitter acetylcholine and a phase of enhanced neuronal excitability. Inhibitors of acetylcholinesterase also promote enhanced electrical brain activity, presumably by increasing the survival of acetylcholine at the synapse. Here we report that there is similar bidirectional modulation of genes that regulate acetylcholine availability after stress and blockade of acetylcholinesterase. These calcium-dependent changes in gene expression coincide with phases of rapid enhancement and delayed depression of neuronal excitability. Both of these phases are mediated by muscarinic acetylcholine receptors. Our results suggest a model in which robust cholinergic stimulation triggers rapid induction of the gene encoding the transcription factor c-Fos. This protein then mediates selective regulatory effects on the long-lasting activities of genes involved in acetylcholine metabolism.

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare “readthrough” acetylcholinesterase variant AChE‐R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE‐R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE‐R in blood and muscle yet leaves unaffected the synaptic variant AChE‐S. Whereas animals treated with placebo or conventional anticho‐linesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA‐targeted therapeutics for chronic cholinergic malfunctioning.—Brenner, T., Hamra‐Amitay, Y., Evron, T., Boneva, N., Seidman, S., Soreq, H. The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis. FASEB J. 17, 214–222 (2003)

Xenopus oocyte microinjection: from gene to protein.

Publisher Summary This chapter discusses the types of experimental protocols, which are exploited in the oocyte expression system. The chapter highlights some novel approaches to Xenopus laevis oocyte microinjection, which are pushing forward the frontiers of molecular neurobiology. As a preliminary step in molecular cloning studies, the microinjection of total poly(A) + mRNA from tissue homogenates into Xenopus oocytes serves to provide evidence for the presence of translatable mRNA encoding the desired polypeptide while establishing the ability of the oocyte to express the biologically active protein. The incalculable contribution of Xenopus oocyte microinjection methods to the rapid progress in molecular and cellular biology can be seen in its application as a translation system; the applications of oocyte microinjection as a heterologous expression system will continue to grow in number and scope..

Anticholinesterases induce multigenic transcriptional feedback response suppressing cholinergic neurotransmission.

Cholinesterase inhibitors (anti-ChEs) include a wide range of therapeutic, agricultural and warfare agents all aimed to inhibit the catalytic activity of the acetylcholine (ACh) hydrolysing enzyme acetylcholinesterase (AChE). In addition to promoting immediate excitation of cholinergic neurotransmission through transient elevation of synaptic ACh levels, anti-ChEs exposure is associated with long-term effects reminiscent of post-traumatic stress disorder. This suggested that exposure to anti-ChEs leads to persistent changes in brain proteins and called for exploring the mechanism(s) through which such changes could occur. For this purpose, we established an in vitro system of perfused, sagittal mouse brain slices which sustains authentic transcriptional responses for over 10 h and enables the study of gene regulation under controlled exposure to anti-ChEs. Slices were exposed to either organophosphate or cabamate anti-ChEs, both of which induced within 10 min excessive overexpression of the mRNA encoding the immediate early response transcription factor c-Fos. Twenty minutes later we noted 8-fold increases over control levels in AChE mRNA, accompanied by a 3-fold decrease in the mRNAs encoding for the ACh synthesizing enzyme choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT). No changes were detected in synaptophysin mRNA levels. These modulations in gene expression paralleled those taking place under in vivo exposure. Of particular concern is the possibility that feedback processes leading to elevated levels of brain AChE may be similarly associated with low-level exposure to common organophosphorous anti-cholinesterases, and lead to long-term deleterious changes in cognitive functions.

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme

Summary1.Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase.2.The recombinant gene product exhibits biochemical traits similar to native “true” acetylcholinesterase as manifested by characteristic substrate inhibition, aKm of 117µM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51.3.The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization.4.The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms.5.In conclusion, the catalytic subunit expressed from the hydrophylic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.

Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle‐specific proteins, among them the acetylcholine‐hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over‐expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell‐associated rHAChE accumulated in myotomes of 2‐and 3‐day‐old embryos within the same sub‐cellular compartments as native Xenopus AChE. NMJs from 3‐day‐old DNA‐injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

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.

Transgenic acetylcholinesterase induces enlargement of murine neuromuscular junctions but leaves spinal cord synapses intact.

Acetylcholinesterase (AChE) produced by spinal cord motoneurons accumulates within axo-dendritic spinal cord synapses. It is also secreted from motoneuron cell bodies, through their axons, into the region of neuromuscular junctions, where it terminates cholinergic neurotransmission. Here we show that transgenic mice expressing human AChE in their spinal cord motoneurons display primarily normal axo-dendritic spinal cord cholinergic synapses in spite of the clear excess of transgenic over host AChE within these synapses. This is in contrast to our recent observation that a modest excess of AChE drastically affects the structure and long-term functioning of neuromuscular junctions in these mice although they express human AChE in their spinal cord, but not muscle. Enlarged muscle endplates with either exaggerated or drastically shortened post-synaptic folds then lead to a progressive neuromotor decline and massive amyotrophy (Andres et al., 1997). These findings demonstrate that excess neuronal AChE may cause distinct effects on spinal cord and neuromuscular synapses and attribute the late-onset neuromotor deterioration observed in AChE transgenic mice to neuromuscular junction abnormalities.

Tissue-specific processing and polarized compartmentalization of clone-produced cholinesterase in microinjected Xenopus oocytes

Summary1.To approach the involvement of tissue-specific elements in the compartmentalization of ubiquitous polymorphic proteins, immunohistochemical methods were used to analyze the localization of butyrylcholinesterase (BuChE) inXenopus oocytes microinjected with synthetic BuChEmRNA alone and in combination with tissue-extracted mRNAs.2.When injected alone BuChEmRNA efficiently directed the synthesis of small membrane-associated accumulations localized principally on the external surface of the oocytes animal pole. Tunicamycin blocked the appearance of such accumulations, suggesting that glycosylation is involved in the transport of nascent BuChE molecules to the oocytes surface. Coinjection with brain or muscle mRNA, but not liver mRNA, facilitated the formation of pronounced, tissue-characteristic BuChE aggregates.3.These findings implicate tissue-specific mRNAs in the assembly of the clone-produced protein and in its nonuniform distribution in the oocyte membrane or extracellular material.