Human recombinant butyrylcholinesterase purified from the milk of transgenic goats interacts with beta-amyloid fibrils and suppresses their formation in vitro.

BACKGROUND: In Alzheimer's disease (AD), brain butyrylcholinesterase (BChE) co-localizes with beta-amyloid (Abeta) fibrils. AIMS: In vitro testing of the significance of this phenomenon to AD progress. METHODS: A thioflavine T (ThT) fluorogenic assay, photo-induced cross-linking and quantifiable electron microscopy served to compare the effect on Abeta fibril formation induced by highly purified recombinant human BChE (rBChE) produced in the milk of transgenic goats with that of serum-derived human BChE. RESULTS: Both proteins at 1:50 and 1:25 ratios to Abeta dose-dependently prolonged the ThT lag time and reduced the apparent rate of Abeta fibril formation compared to Abeta alone. Photo-induced cross-linking tests showed that rBChE prolonged the persistence of amyloid dimers, trimers and tetramers in solution, whereas Abeta alone facilitated precipitation of such multimers from solution. Transmission electron microscopy showed that rBChE at 1:100 to Abeta prevented the formation of larger, over 150-nm-long, Abeta fibrils and reduced fibril branching compared to Abeta alone as quantified by macro programming of Image Pro Plus software. CONCLUSION: Our findings demonstrate that rBChE interacts with Abeta fibrils and can attenuate their formation, extension and branching, suggesting further tests of rBChE, with unlimited supply and no associated health risks, as a therapeutic agent for delaying the formation of amyloid toxic oligomers in AD patients.

Acetylcholinesterase-R increases germ cell apoptosis but enhances sperm motility.

Changes in protein subdomains through alternative splicing often modify protein-protein interactions, altering biological processes. A relevant example is that of the stress-induced up-regulation of the acetylcholinesterase (AChE-R) splice variant, a common response in various tissues. In germ cells of male transgenic TgR mice, AChE-R excess associates with reduced sperm differentiation and sperm counts. To explore the mechanism(s) by which AChE-R up-regulation affects spermatogenesis, we identified AChE-R's protein partners through a yeast two-hybrid screen. In meiotic spermatocytes from TgR mice, we detected AChE-R interaction with the scaffold protein RACK1 and elevated apoptosis. This correlated with reduced scavenging by RACK1 of the pro-apoptotic TAp73, an outcome compatible with the increased apoptosis. In contrast, at later stages in sperm development, AChE-R's interaction with the glycolytic enzyme enolase-alpha elevates enolase activity. In transfected cells, enforced AChE-R excess increased glucose uptake and adenosine tri-phosphate (ATP) levels. Correspondingly, TgR sperm cells display elevated ATP levels, mitochondrial hyperactivity and increased motility. In human donors' sperm, we found direct association of sperm motility with AChE-R expression. Interchanging interactions with RACK1 and enolase-alpha may hence enable AChE-R to affect both sperm differentiation and function by participating in independent cellular pathways.

Acetylcholinesterase/C terminal binding protein interactions modify Ikaros functions, causing T lymphopenia.

Hematological changes induced by various stress stimuli are accompanied by replacement of the primary acetylcholinesterase (AChE) 3' splice variant acetylcholinesterase-S (AChE-S) with the myelopoietic acetylcholinesterase-R (AChE-R) variant. To search for putative acetylcholinesterase-S interactions with hematopoietic pathways, we employed a yeast two-hybrid screen. The transcriptional co-repressor C-terminal binding protein (CtBP) was identified as a protein partner of the AChE-S C terminus. In erythroleukemic K562 cells, AChE-S displayed nuclear colocalization and physical interaction with CtBP. Furthermore, co-transfected AChE-S reduced the co-repressive effect of CtBP over the hematopoietic transcription factor, Ikaros. In transgenic mice, overexpressed human (h) AChE-S mRNA induced selective bone marrow upregulation of Ikaros while suppressing FOG, another transcriptional partner of CtBP. Transgenic bone marrow cells showed a correspondingly elevated potential for producing progenitor colonies, compared with controls, while peripheral blood showed increased erythrocyte counts as opposed to reduced platelets, granulocytes and T lymphocytes. AChE's 3' alternative splicing, and the corresponding changes in AChE-S/CtBP interactions, thus emerge as being actively involved in controlling hematopoiesis and the potential for modulating immune functions, supporting reports on malfunctioning immune reactions under impaired splice site selection.

SC35 promotes sustainable stress-induced alternative splicing of neuronal acetylcholinesterase mRNA.

Long-lasting alternative splicing of neuronal acetylcholinesterase (AChE) pre-mRNA occurs during neuronal development and following stress, altering synaptic properties. To explore the corresponding molecular events, we sought to identify mRNAs encoding for abundant splicing factors in the prefrontal cortex (PFC) following stress. Here we show elevated levels of the splicing factor SC35 in stressed as compared with naïve mice. In cotransfections of COS-1 and HEK293 cells with an AChE minigene allowing 3' splice variations, SC35 facilitated a shift from the primary AChE-S to the stress-induced AChE-R variant, while ASF/SF2 caused the opposite effect. Transfection with chimeric constructs comprising of SC35 and ASF/SF2 RRM/RS domains identified the SC35 RRM as responsible for AChE mRNA's alternative splicing. In poststress PFC neurons, increased SC35 mRNA and protein levels coincided with selective increase in AChE-R mRNA. In the developing mouse embryo, cortical progenitor cells in the ventricular zone displayed transient SC35 elevation concomitant with dominance of AChE-R over AChE-S mRNA. Finally, transgenic mice overexpressing human AChE-R, but not those overexpressing AChE-S, showed significant elevation in neuronal SC35 levels, suggesting a reciprocal reinforcement process. Together, these findings point to an interactive relationship of SC35 with cholinergic signals in the long-lasting consequences of stress on nervous system plasticity and development.