Neuronal overexpression of ‘readthrough’ acetylcholinesterase is associated with antisense-suppressible behavioral impairments

Abstract

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.

Comparative developmental neurotoxicity of organophosphates in vivo: Transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems

“…Indeed, using a reverse transcriptasepolymerase chain reaction approach, we recently identified selective upregulation of the acetylcholinesterase synaptic splice variant, AChE-S, by the same DZN treatment used here, whereas CPF was ineffective. Because AChE-S is specifically associated with neurotoxic endpoints [21,71,85,102], our negative findings for global ache expression may be misleading in that effects on a specific splice variant may be masked by dilution of the mRNA from this subtype with unaffected variants. This points out an additional limitation of the microarray approach in situations where probes for multiple splice variants are not present on the array.…”

Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation

“…To study the implications of AChE-R mRNA upregulation for fear conditioning, we employed mEN101, an antisense oligonucleotide inducing murine AChE-R mRNA downregulation. 15 When intracerebroventricularly (i.c.v.) injected 15 min before immobilization, mEN101 selectively limited the stress-induced accumulation of AChE-R mRNA and protein to less than half of its full scale (Figures 2a and b and 3).…”

“…14 Unlike the abundant AChE-S 'synaptic' variant, the stress-induced AChE-R possesses a hydrophilic C-terminus that is expected to be incapable of supporting membrane adherence. Neuronal AChE-R accumulation is accompanied by long-lasting hyperexcitation of glutamatergic activity 15 and prolonged conflict behavior. 16 To explore the possibility that changes in alternative splicing are critically involved in the stress-induced consolidation of fear memory, we tested the potential interrelationship between neuronal overproduction of the 'readthrough' AChE-R variant, 17 stress-enhanced fear memory and facilitated long-term potentiation (LTP) in the hippocampus.…”

“…15 InvEN101 is the inverted sequence (negative control oligodeoxynucleotide). A measure of 5 mM oligodeoxynucleotides was combined with 13 mM of the lipophilic transfection reagent DOTAP (Boehringer Mannheim, Germany) in an artificial cerebrospinal fluid (aCSF) and incubated for 15 min at 371C prior to injection (of 25 ng in 1 ml).…”

Interaction of “readthrough” acetylcholinesterase with RACK1 and PKCβII correlates with intensified fear-induced conflict behavior

“…3A). EN101, a 2Ј-oxymethylated antisense oligonucleotide, which successfully suppresses stress-induced increases in locomotor activity (33,34), was administered i.p. and served to study the physiological significance of AChE-R-RACK1-PKC␤II interactions.…”