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Genetic Studies
Project Summary

Title: Transgenic Engineering of Cholinesterases: Tools for Exploring Cholinergic Responses
Synopsis: The study looks at the various chemical enzymes that exists in the body in order to find a better means of protecting cells from the harmful effects of organophosphates.
Overall Project Objective: To assess the potential of transgenic cholinesterases to protect organisms against anticholinesterase neurotoxicity.
Status/Results to Date: We have constructed and expressed a variety of catalytically active and inactive variants of AChE in Xenopus oocytes and embryos, identified neurite growth-promoting activities of these variants and shown this activity to be independent of acetylcholine hydrolysis. These constructs were then used to establish new lines of transgenic mice which express the transgenes. DNA encoding an antisense RNA targeted against rodent AChE was used to prove functional redundancy in neuritogenesis between AChE and the AChE-homologous protein neuroligin. In normal mice, we found that both acute psychological stress and Ops induce AChE overproduction, conferring delayed protection from AChE inhibitors. Next, the protective activity of the different transgenes to OP poisons will be tested, together with the long-term performance of these mice in tests of cognition and neuromotor function. DNA samples are also being collected from human subjects presenting hypersensitivity to acetylcholinesterases used in medical or agricultural settings and searching for possible allelic variations in either the AChE or BCHE gene which might be correlated with adverse reactions to these compounds. Identified a single point mutation in the human BCHE gene coding sequence and a rare polymorphism in the human ACHE gene upstream promoter region that may confer hypersensitivity to a variety of cholinesterase inhibitors.
Project:DoD-51
Agency:Department Of Defense
Location:Hebrew University of Jerusalem
P.I. Name:Hermona Soreq, Ph. D.
Research Type:Mechanistic
Research Focus:Chemical Weapons
Focus Category:Genetic Studies
Status:Complete
Study Start Date:October 01,1996
Estimated Completion Date:February 28,2000
Specific Aims: Our previous work has demonstrated that overexpression of a specific alternative variant of the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) in brain protects transgenic mice against the acute toxicity of organophosphate (OP) poisons. However, we also observed that chronic overexpression of AChE in the central nervous system (CNS) may promote delayed impairments in cognitive and neuromotor function. Our objectives, therefore, are to determine the specific cholinesterase isoforms and mode(s) of expression conferring the greatest level of protection against OP poisoning with the least side effects. To this end, we aim to develop animal models for testing the protective value and potential side-effects of transgenic overexpression of various human cholinesterases. In addition, we shall search for genetic factors conferring variability in the susceptibility of individuals to OP intoxication.
Methodology: To study the protective effects of overexpressed AChE on the nervous system we established transgenic mice carrying two copies of the human gene encoding the synaptic form of AChE. These mice display elevated levels of AChE in central cholinergic neurons normally expressing this enzyme and reduced sensitivity to cholinergic intoxication. However, despite apparently normal embryonic and early post-natal development, ACHE-transgenic mice exhibit delayed-onset, progressive impairments in central cognitive functions such as learning and memory, and in neuromotor function. To determine the protective value of alternative AChE isoforms with different subcellular and tissue-specific patterns of expression, and to dissect the mechanism(s) through which excess AChE mediates late onset neuropathologies, we are establishing additional lines of transgenic mice expressing soluble or inactive forms of the human protein. Massive overexpressions was observed in muscle and milk, but very limited excess in brain. However, clear neuropathology could be detected in the somatosensory cortex of mice expressing either inactive or active transgenic synaptic AchE, but much less so with the soluble enzyme. This demonstrates that the long term effects of overexpression are not solely dependent on the catalytic activity of the enzyme, but depend on specific alternative C-terminal peptide domains which exert more active roles in mediating neurodegenerative processes. To investigate possible genetic polymorphisms affecting sensitivity to anticholinesterases we are studying upstream regulatory sequences in the human ACHE gene promoter .
Most Recent Publications:

Sternfeld M, Ming G, Song H, Sela K, Timberg R, Poo M, Soreq H. Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini. The Journal of Neuroscience, 18(4):1240-9, Feb 1998. Abstract

Andres C, Beeri R, Friedman A, Lev-Lehman E, Henis S, Timberg R, Shani M, Soreq H. Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin 1b gene expression followed by late onset neuromotor deterioration. Pro. Natl Academy of Sciences, 22;94(15):8173-8, Jul 1997. Abstract

Kaufer D, Friedman A, Seidman S, Soreq H. Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature, 28;393(6683):373-7, May 1998. Abstract

Kaufer D, Friedman A, Seidman S, Soreq H. Anticholinesterases induce multigenic transcriptional feedback response suppressing cholinergic neuro transmission. Chem Biol Interact, 14;119-120:349-60, May 1999. Abstract

Beeri R, Le Novere MR, Huberman T, Grauer E, Changeux JP, Soreq H. Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice. Journal of Neurochem, 69(6):2441-51, Dec 1997. Abstract

Grifman M, Galyam N, Seidman S, Soreq H. Functional redundancy of acetylcholinesterase and neuroligin in mamalian neuritogenesis. Pro. Natl Academy of Sciences, 10;95(23):13935-40, Nov 1998. Abstract

Grisaru D, Lev-Lehman E, Shapira M, Chikin E, Lessing JB, Eldor A, Eckstein F, Soreq H. Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3 alternative splicing variant of acetylcholinesterase. Molec Cell Biol, 19(1):788-95, Jan 1999. Abstract

Sternfeld M, Patrick JD, Soreq H. Position effect variegations and brain-specific silencing in transgenic mice overexpressing human acetylcholinesterase variants. Journal of Physiology (Paris), 92(3-4):249-55, Jun-Aug, 1998. Abstract

Andres C, Seidman S, Beeri R, Timberg R, Soreq H. Transgenic acetylcholinesterase induces enlargement of murine neuromuscular junctions but leaves spinal cord synapses intact. Neurochem Int, 32(5-6):449-56, May-Jun, 1998. Abstract