Contributions of selective knockout studies to understanding cholinesterase disposition and function

Abstract

The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.

Fig.1

The short span of the ACHE gene is shown contained within an 8.5kb Hind III restriction fragment of mouse genomic DNA [1]. Consistent to all forms of AChE is the invariant splicing of exons 2, 3 and 4 which produces the message which encodes the catalytic core of the enzyme. The AChE gene includes three alternative 3′ end splices for AChE mRNA. Reading through the 3′ end of exon 4 encodes mRNA for soluble AChE monomers. The splice to exon 5 produces mRNA that encodes glycophospholipid linked AChE that is found on the surface of red cells and platelets. The splice to exon 6 produces mRNA for the most commonly studied form of the enzyme which is found in brain and muscle. Multimers of tetramers of the AChE subunit translated from exon 6 spliced mRNA are tethered in place by ColQ and PRiMA subunits in brain and muscle.

Fig. 2

AChE activity in tissues and blood of the WT mouse (mixed strain, WT littermates of knockout mice). Tissue AChE activity is reported as U AChE /g tissue. Serum is reported as U AChE/ml serum, platelets and red cell ghost values are reported as U AChE/ml blood.