To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.
Molecular origin(s) of the diverse behavioral responses to anticholinesterases were explored in behaviorally impaired transgenic (Tg) FVB/N mice expressing synaptic human acetylcholinesterase (hAChE-S). Untreated hAChE-S Tg, unlike naïve FVB/N mice, presented variably intense neuronal overexpression of the alternatively spliced, stress-induced mouse 'readthrough' mAChE-R mRNA. Both strains displayed similar diurnal patterns of locomotor activity that were impaired 3 days after a day-to-night switch. However, hAChE-S Tg, but not FVB/N mice responded to the circadian switch with irregular, diverse bursts of increased locomotor activity. In social recognition tests, controls displayed short-term recognition, reflected by decreased exploration of a familiar, compared to a novel juvenile conspecific as well as inverse correlation between social recognition and cortical and hippocampal AChE specific activities. In contrast, transgenics presented poor recognition, retrievable by tetrahydroaminoacridine (tacrine, 1.5 mg kg −1 ). Tacrine's effect was short-lived (Ͻ40 min), suggesting its effect was overcome by anticholinesterase-induced overproduction of mAChE-R. Consistent with this hypothesis, antisense oligonucleotides (two daily intracerebroventricular injections of 25 ng) arrested mAChE-R synthesis, selectively reduced mAChE-R levels and afforded an extended (Ͼ24 h) suppression of the abnormal social recognition pattern in transgenics. Efficacy of antisense treatment was directly correlated with AChE-R levels and the severity of the impaired phenotype, being most apparent in transgenics presenting highly abnormal pretreatment behavior. These findings demonstrate that neuronal AChE-R overproduction is involved in various behavioral impairments and anticholinesterase responses, and point to the antisense strategy as a potential approach for re-establishing cholinergic balance.
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