Defects in pre-mRNA processing as causes of and predisposition to diseases.

Humans possess a surprisingly low number of genes and intensively use pre-mRNA splicing to achieve the high molecular complexity needed to sustain normal body functions and facilitate responses to altered conditions. Because hundreds of thousands of proteins are generated by 25,000 to 40,000 genes, pre-mRNA processing events are highly important for the regulation of human gene expression. Both inherited and acquired defects in pre-mRNA processing are increasingly recognized as causes of human diseases, and almost all pre-mRNA processing events are controlled by a combination of protein factors.

Complex regulation of acetylcholinesterase gene expression in human brain tumors.

To study the regulation of acetylcholinesterase (AChE) gene expression in human brain tumors, 3' splice variants of AChE mRNA and potentially relevant transcription factor mRNAs were labeled in primary astrocytomas and melanomas. AChE-S and AChE-R mRNA, as well as Runx1/AML1 mRNA accumulated in astrocytomas in correlation with tumor aggressiveness, but neither HNF3beta nor c-fos mRNA was observed in melanoma and astrocytomas. Immunohistochemistry demonstrated nuclear Runx1/AML1 and cellular AChE-S and AChE-R in melanomas, however, only AChE-S, and not the secreted AChE-R variant, was retained in astrocyte tumor cells.

Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage display library.

Current Alzheimer's disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study this phenomenon we developed monoclonal antibodies to acetylcholinesterase synaptic peptide (ASP), a synthetic peptide with the C-terminal sequence unique to the human synaptic variant AChE-S.

The stress-associated acetylcholinesterase variant AChE-R is expressed in human CD34(+) hematopoietic progenitors and its C-terminal peptide ARP promotes their proliferation.

Objective: Hematopoietic stress responses involve increases in leukocyte and platelet counts, implying the existence of stress responsive factors that modulate hematopoiesis. Acetylcholinesterase (AChE) is expressed in mammalian neurons and hematopoietic cells. In brain, it responds to stress by mRNA overexpression and alternative splicing, yielding the rare stress-associated "readthrough" AChE-R variant protein.

Neuronal overexpression of "readthrough" acetylcholinesterase is associated with antisense-suppressible behavioral impairments.

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.

Sustained cholinesterase inhibition in AD patients receiving rivastigmine for 12 months.

Objective: To study the long-term dual inhibitory effects of rivastigmine on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in patients with AD.

Methods: Eleven patients with mild AD received rivastigmine for 12 months. Cholinesterase (ChE) activities in the CSF and plasma were assessed colorimetrically.

Transcriptional regulation of erythropoiesis. Fine tuning of combinatorial multi-domain elements.

Haematopoiesis, the differentiation of haematopoietic stem cells and progenitors into various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Commitment of pluripotent haematopoietic stem cells to the erythroid lineage induces erythropoiesis, the production of red blood cells. This process involves a concerted progression through an erythroid burst forming unit (BFU-E), an erythroid colony forming unit (CFU-E), proerythroblast and an erythroblast.

Upregulation of neuronal nicotinic receptor subunits alpha4, beta2, and alpha7 in transgenic mice overexpressing human acetylcholinesterase.

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits alpha4, beta2, and alpha7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [3H]epibatidine and [125I]alphabungarotoxin binding in the cortex and the caudate putamen of these mice.

Genomic dissection reveals locus response to stress for mammalian acetylcholinesterase.

The mammalian acetylcholinesterase (ACHE) locus was investigated using computational predictive methods and experiments of reverse transcription polymerase chain reaction (RT-PCR). Computational analysis identified two genes downstream to ACHE, an inversely oriented arsenite resistance gene homologue (ARS), and a novel previously unidentified gene (PIX), co-oriented with ACHE. Experimental evidence shows coregulation of murine ACHE and ARS following confined swim, indicating coordinated locus response to stress, that is possibly mediated by altered cholinergic neurotransmission.

Frequent blood-brain barrier disruption in the human cerebral cortex.

1. The blood-brain barrier (BBB) protects the brain from circulating xenobiotic agents. The pathophysiology, time span, spatial pattern, and pathophysiological consequences of BBB disruptions are not known.

Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity.

To explore neuronal mechanisms underlying long-term consequences of stress, we studied stress-induced changes in the neuritic translocation of acetylcholinesterase (AChE) splice variants. Under normal conditions, we found the synaptic AChE-S mRNA and protein in neurites. Corticosterone, anticholinesterases, and forced swim, each facilitated a rapid (minutes), yet long-lasting (weeks), shift from AChE-S to the normally rare AChE-R mRNA, promoted AChE-R mRNA translocation into neurites, and induced enzyme secretion.

Runx1/AML1 in leukemia: disrupted association with diverse protein partners.

Runx1/AML1, a chromosome 21q22 hematopoietic regulator, is frequently translocated in leukemia. Its protein product, a relatively weak transcriptional activator, becomes an effective transcriptional enhancer or repressor, when co-operating with transcriptional co-activators or co-repressors. Runx1/AML1 association with its partners is disrupted in leukemia.

Changes in neuronal acetylcholinesterase gene expression and division of labor in honey bee colonies.

Division of labor in honey bee colonies is highlighted by adult bees making a transition at 2-3 wk of age from working in the hive to foraging for nectar and pollen outside. This behavioral development involves acquisition of new tasks that may require advanced learning capabilities. Because acetylcholinesterase (AChE) hydrolyzes acetylcholine, a major neurotransmitter associated with learning in the insect brain, we searched for changes in AChE expression in the brain during bee behavioral development.

Expression of recombinant human acetylcholinesterase in transgenic tomato plants.

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants.

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility.

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress-related infertility are not yet known. Overexpression of the "readthrough" variant of acetylcholinesterase (AChE-R) is involved in the cellular stress response in a variety of mammalian tissues.

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

Background: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses.

Materials And Methods: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin.

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase.

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice.

Acetylcholinesterase--new roles for an old actor.

The discovery of the first neurotransmitter--acetylcholine--was soon followed by the discovery of its hydrolysing enzyme, acetylcholinesterase. The role of acetylcholinesterase in terminating acetylcholine-mediated neurotransmission made it the focus of intense research for much of the past century. But the complexity of acetylcholinesterase gene regulation and recent evidence for some of the long-suspected 'non-classical' actions of this enzyme have more recently driven a profound revolution in acetylcholinesterase research.

Complex host cell responses to antisense suppression of ACHE gene expression.

3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2'-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.02-100 nM.

Antisense prevention of neuronal damages following head injury in mice.

Closed head injury (CHI) is an important cause of death among young adults and a prominent risk factor for nonfamilial Alzheimer's disease. Emergency intervention following CHI should therefore strive to improve survival, promote recovery, and prevent delayed neuropathologies. We employed high-resolution nonradioactive in situ hybridization to determine whether a single intracerebro-ventricular injection of 500 ng 2'-O-methyl RNA-capped antisense oligonucleotide (AS-ODN) against acetylcholinesterase (AChE) mRNA blocks overexpression of the stress-related readthrough AChE (AChE-R) mRNA splicing variant in head-injured mice.

Anti-sense approach to anticholinesterase therapeutics.

The acetylcholine-hydrolyzing enzyme, acetylcholinesterase, is the molecular target of approved drugs for Alzheimer's disease and myasthenia gravis. However, recent data implicate AChE splicing variants in the etiology of complex diseases such as AD and MG. Despite the large arsenal of anti-AChE drugs, therapeutic inhibitors are primarily targeted towards an active site shared by all variants.

Genomic and transcriptional characterization of the human ACHE locus: complex involvement with acquired and inherited diseases.

Background: Abnormal levels of the acetylcholinesterase enzyme or aberrations involving the long arm of chromosome 7, harboring the ACHE gene at 7q22, occur in various diseases such as Alzheimer's, Parkinson's, and leukemias. However, the cause(s) of these abnormalities are still unknown.

Objective: To search for the genomic elements and transcriptional processes controlling ACHE gene expression and the plausible stability of its locus, by isolating, sequencing and characterizing the human (h)ACHE locus and its mRNA products.

Excess "read-through" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates.

Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The "read-through" variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the "synaptic" AChE variant, AChE-S.

Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.

Background: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized.