Readthrough Acetylcholinesterase Research Articles

Increased Active OMI/HTRA2 Serine Protease Displays a Positive Correlation with Cholinergic Alterations in the Alzheimer\u2019s Disease Brain

OMI/HTRA2 (high-temperature requirement serine protease A2) is a mitochondrial serine protease involved in several cellular processes, including autophagy, chaperone activity, and apoptosis. Few studies on the role of OMI/HTRA2 in Alzheimer’s disease (AD) are available, but none on its relationship with the cholinergic system and neurotrophic factors as well as other AD-related proteins. In this study, immunohistochemical analyses revealed that AD patients had a higher cytosolic distribution of OMI/HTRA2 protein compared to controls. Quantitative analyses on brain extracts indicated a significant increase in the active form of OMI/HTRA2 in the AD brain. Activated OMI/HTRA2 protein positively correlated with stress-associated read-through acetylcholinesterase activity. In addition, α7 nicotinic acetylcholine receptor gene expression, a receptor also known to be localized on the outer membrane of mitochondria, showed a strong correlation with OMI/HTRA2 gene expression in three different brain regions. Interestingly, the activated OMI/HTRA2 levels also correlated with the activity of the acetylcholine-biosynthesizing enzyme, choline acetyltransferase (ChAT); with levels of the neurotrophic factors, NGF and BDNF; with levels of the soluble fragments of amyloid precursor protein (APP); and with gene expression of the microtubule-associated protein tau in the examined brain regions. Overall, the results demonstrate increased levels of the mitochondrial serine protease OMI/HTRA2, and a coherent pattern of association between the activated form of OMI/HTRA2 and several key proteins involved in AD pathology. In this paper, we propose a new hypothetical model to highlight the importance and needs of further investigation on the role of OMI/HTRA2 in the mitochondrial function and AD.

Increased Expression of Readthrough Acetylcholinesterase Variants in the Brains of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to controls. Similar increases were detected by western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties that are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine.

Strain dependent effects of conditioned fear in adult C57Bl/6 and Balb/C mice following postnatal exposure to chlorpyrifos: relation to expression of brain acetylcholinesterase mRNA.

Following reports of emotional psychopathology in children and adults exposed to organophosphates, the effects of postnatal chlorpyrifos (CPF) on fear-conditioning and depression-like behaviors were tested in adult mice. Concomitant changes in expression of mRNA for synaptic and soluble splice variants of acetylcholinesterase (AChE) were examined in mouse pups and adults of the Balb/C and C57Bl/6 (B6) strains, which differ in their behavioral and hormonal stress response. Mice were injected subcutaneously with 1 mg/kg CPF on postnatal days 4–10 and tested as adults for conditioned fear, sucrose preference, and forced swim. Acetylcholinesterase activity was assessed in the brains of pups on the first and last day of treatment. Expression of soluble and synaptic AChE mRNA was assessed in brains of treated pups and fear-conditioned adults using real-time PCR. Adult Balb/C mice exposed postnatally to CPF showed exacerbated fear-conditioning and impaired active avoidance. Adult B6 mice exposed postnatally to CPF showed a more specific fear response to tones and less freezing in the inter-tone intervals, in contrast to the vehicle-pretreated mice. Chlorpyrifos also attenuated sweet preference and enhanced climbing in the forced swim test. Chlorpyrifos-treated mice had increased expression of both synaptic and readthrough AChE transcripts in the hippocampus of Balb/C mice and decreased expression in the amygdala following fear-conditioning. In conclusion, postnatal CPF had long-term effects on fear and depression, as well as on expression of AChE mRNA. These changes may be related to alteration in the interaction between hippocampus and amygdala in regulating negative emotions.

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Postnatal diisopropylfluorophosphate enhances conditioned vigilance in adult BALB/c and C57BL/6 mice and alters expression of acetylcholinesterase splice variants.

The long-term effects of postnatal exposure to an organophosphate substance diisopropylfluorophosphate (DFP) were examined on fear conditioning in adult mice. Immediate and long-term changes in the expression of synaptic acetylcholinesterase (AChE-S) and readthrough acetylcholinesterase (AChE-R) transcripts were explored, in view of reports relating expression of these splice variants to stress and anxiety. BALB/c and C57BL/6 mice were injected daily, on postnatal days 4-10, with 1 mg/kg of DFP or saline and tested as adults for cued and contextual freezing and scanning. Real-time PCR was used to investigate expression of the rare AChE-R and AChE-S mRNA postnatally and in fear-conditioned adults. DFP-pretreated male mice showed increased conditioned cued scanning and both male and female DFP-treated mice showed enhanced contextual scanning. DFP abolished the stress-induced increase in AChE transcript expression in BALB/c but not in C57BL/6 mice. A significant correlation was found between expression of AChE-S and AChE-R transcripts in the hippocampus and scanning behavior, but this was apparently unrelated to DFP treatment. The enhanced conditioned scanning in adults, following postnatal exposure to DFP, suggests long-term effects on the risk for anxiety disorders. The altered expression of AChE splice variant transcripts in the two strains did not account for the behavioral deficits, which were observed in both BALB/c and C57BL/6 strains.

Pathophysiological amyloid concentrations induce sustained upregulation of readthrough acetylcholinesterase mediating anti-apoptotic effects

Cholinergically differentiated SH-SY5Y neuroblastoma cells were treated with a pathophysiologically relevant, low (300nM), and a high (3μM) dose of amyloid beta 1-42 (Abeta) or 42-1 (revAbeta). At early (1 and 4h) and late (24h) time points, the pro- and anti-apoptotic factors – caspase-3 and p53, and B-cell lymphoma 2 protein (Bcl-2), respectively – were assessed together with lactate dehydrogenase (LDH) release as measure of cell viability and ATP levels as marker of mitochondrial activity. The low peptide dose significantly increased Bcl-2 and, time-delayed, caspase-3 and ATP levels, but barely impacted on LDH release, while the high concentration remarkably depressed Bcl-2 levels, depleted ATP and led to increased LDH release. We also monitored acetylcholinesterase (AChE) enzymatic activity and splice variant levels (tailed and readthrough AChE; AChE-T and AChE-R), and assessed choline acetyltransferase (ChAT) and high-affinity choline uptake (HACU). The low Abeta concentration drastically upregulated AChE-R and increased both ChAT and HACU, while the high dose caused cholinergic toxicity. We believe this study offers the first insight into the highly concentration-dependent effects of Abeta on cholinergic dynamics. In particular, it highlights the rescuing role of AChE-R as being, together with mitochondrial activity, involved in cholinergic adaptation to low doses of Abeta.

Readthrough Acetylcholinesterase Is Increased in Human Liver Cirrhosis

Background & AimsThere have been many studies on plasma butyrylcholinesterase in liver dysfunction. However, no data is available about acetylcholinesterase in human cirrhosis, although profound changes have been described in cirrhotic rat models.MethodsHuman serum and liver acetylcholinesterase and its molecular forms were determined enzymatically, after butyrylcholinesterase immunodepletion. The distinct species of acetylcholinesterase, with a distinct C-terminus, were determined by western blotting, and the level of liver transcripts by real-time PCR. Liver acetylcholinesterase was also evaluated by immunocytochemistry.ResultsIn patients with liver cirrhosis, the activity of plasma acetylcholinesterase (rich in light species), appeared to be apparently unaffected. However, the levels of the soluble readthrough (R) acetylcholinesterase form, an acetylcholinesterase species usually associated with stress and pathology, was increased compared to controls. Human liver acetylcholinesterase activity levels were also unchanged, but protein levels of the acetylcholinesterase-R and other acetylcholinesterase subunit species were increased in the cirrhotic liver. This increase in acetylcholinesterase protein expression in the cirrhotic liver was confirmed by PCR analysis. Immunohistological examination confirmed that acetylcholinesterase immunoreactivity is increased in parenchymal cells of the cirrhotic liver.ConclusionsWe demonstrate significant changes in acetylcholinesterase at the protein and mRNA levels in liver cirrhosis, with no difference in enzymatic activity. The altered expression of acetylcholinesterase protein may reflect changes in its pathophysiological role.

Dramatic increase in readthrough acetylcholinesterase in a cellular model of oxidative stress

Moderate, transient oxidative stress is achieved in SH-SY5Y cells using tertiary butylhydroperoxide as oxidant. Over a recovery period of 24 h, the enzymatic activity and protein levels of the acetylcholinesterase (AChE) splice variants tailed AChE (AChE-T) and readthrough AChE (AChE-R) are monitored. Their time-dependent correlation to pro- and anti-apoptotic factors, namely caspase 3 and Bcl-2, respectively, as well as lactate dehydrogenase release as a measure of cell viability is assessed. A distinctly different expression pattern of AChE-T as compared with AChE-R is recorded, in that AChE-T shows only a very slight increase over a 6 h time period. In contrast, AChE-R rises continuously during the recovery period, reaching peak intracellular levels that are up to six times higher than control levels 3-4 h post-stress, and is released from cells in substantial amounts. Moreover, anti-apoptotic Bcl-2 increases significantly, peaking 2-3 h after this AChE-R peak has occurred. We believe this study presents the first work that demonstrates - without relying on techniques of over-expression - the time-dependent correlation between apoptotic processes and related rescue mechanisms involving AChE isoforms in a neuronal cell line.

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology

Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the 'Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid beta (Abeta) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Abeta oligomers and fibrils and abolished Abeta toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Abeta-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic events.

The effect of the readthrough acetylcholinesterase variant (AChE-R) on uterine muscle and leiomyomas

Acetylcholine signaling and acetylcholinesterase (AChE) function(s) are pivotal elements in muscle development. The effects of the stimulus-dependent readthrough AChE variant, AChE-R, on leiomyomas and normal myometrium proliferation were assessed in vivo and in vitro. Histological preparations and cell cultures therefrom were obtained during hysterectomies or myomectomies and included both the leiomyoma sample and the adjacent normal uterine muscle as control. In situ hybridization procedures were performed using AChE cRNA probes complementary to the human AChE-R transcript. Antibodies against the AChE-R variant served for immunohistochemical staining. To determine the biological function of AChE-R on the uterine muscle cell cultures, we used a synthetic peptide representing the potentially cleavable morphogenically active C-terminus of AChE-R (ARP). Cell proliferation was assessed using the incorporation of 5'-bromo-2-deoxyuridine (BrDU). Leiomyomas expressed an excess of AChE-R mRNA and the AChE-R protein compared with the normal myometrium. Cell cultures originating from leiomyomas proliferated significantly faster than cultures from the adjacent myometrium (P = 0.027 at BrDU incorporation). Addition of ARP (2-200 nM) caused a dose-dependent decrease in the proliferation of cell cultures from both leiomyomas and the myometrium. The effect on the myometrium reached statistical significance (at 20 and 200 nM, P = 0.02), whereas the variability of the rapidly proliferating primary cultures was high and precluded statistical significance in the leiomyoma cultures. AChE-R is involved in the proliferation of the myometrium. The inhibitory effect of ARP on the myometrium may suggest a future therapeutic role of ARP.

Readthrough acetylcholinesterase in inflammation-associated neuropathies

The cholinergic control over inflammatory reactions calls for deciphering the corresponding protein partners. An example is blood–nerve barrier disruption allowing penetration of inflammatory factors, which is notably involved in various neuropathies due to yet unknown molecular mechanism(s). In rats, lipopolysaccharide (LPS) administration followed by intra-neural (i.n.) saline injection inducing a focal blood–nerve disruption leads to systemic inflammatory reaction accompanied by transient conduction impairment in the sciatic nerve. Here, we provide evidence compatible with the hypothesis that ARP, the naturally cleavable C-terminal peptide of the stress-induced “readthrough” acetylcholinesterase variant (AChE-R), is causally involved in the emergence of this LPS-induced conduction impairment. Intra-neural injection to naïve rats of conditioned medium from cultured splenocytes exposed to LPS in vitro (reactive splenocyte medium) induced a transient conduction impairment that was accompanied by facilitated accumulation of cleaved intra-neural ARP. Protein kinase C (PKC) βII, known to interact with ARP, was significantly elevated in the LPS-exposed sciatic nerve preparations. Moreover, direct i.n. injection of synthetic ARP30, bearing the mouse AChE-R C-terminal sequence, similarly induced PKCβII expression and conduction impairment. The induction of neural conduction impairment by ARP, possibly through its interaction with PKCβII, suggests a role for AChE-R expression in inflammation-associated neuropathies.

Readthrough Acetylcholinesterase: A Multifaceted Inducer of Stress Reactions

Stress insults induce hyperexcitation of cholinergic circuits, both peripherally in the sympathetic pathway (Tracey, 2002) and at the central nervous system (CNS) (Sapolsky, 1996). This reaction can serve to ensure survival but might also entail a risk to the hyperactivated neurons. Consequent changes in the expression of a series of proteins related to acetylcholine (ACh) metabolism might protect the organism from the potentially detrimental effects of this increase in ACh. Of particular interest among these effects is the induction by alternative splicing of the alternative, usually rare, readthrough variant of acetylcholinesterase (AChE), AChE-R. AChE-R is one of the first proteins conveying the signal that the organism has entered a state of alert. Viewing AChER as a stress signal can therefore serve to answer the question "How do we expect a stress signal to operate"? This facilitates the generation of hypotheses regarding the triggering of such signals and the effects it exerts at the molecular, cellular, and physiological levels.

Readthrough Acetylcholinesterase Expression Remains Minor After Stress or Exposure to Inhibitors

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis.

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare "readthrough" acetylcholinesterase variant AChE-R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE-R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE-R in blood and muscle yet leaves unaffected the synaptic variant AChE-S. Whereas animals treated with placebo or conventional anticholinesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA-targeted therapeutics for chronic cholinergic malfunctioning.