Mirta Grifman

In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzymes affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Manipulations of ACHE gene expression suggest non-catalytic involvement of acetylcholinesterase in the functioning of mammalian photoreceptors but not in retinal degeneration

To explore role(s) of acetylcholinesterase (AChE) in functioning and diseased photoreceptors, we studied normal (rd/+) and degenerating (rd/rd) murine retinas. All retinal neurons, expressed AChEmRNA throughout fetal development. AChE and c-Fos mRNAs peaked at post-natal days 10-12, when apoptosis of rd/rd photoreceptors begins. Moreover, c-Fos and AChEmRNA were co-overexpressed in rd/rd mice producing transgenic human (h), and host (m) AChE, but not in rd/+ mice. However, mAChE overexpression also occurred in transgenics expressing human serum albumin. Drastic variations in AChE catalytic activity were ineffective during development. Neither transgenic excess nor diisopropylfluorophosphonate (DFP) inhibition (80%) affected the rd phenotype; nor did DFP exposure induce photoreceptor degeneration or affect other key cholinergic proteins in rd/+ mice, unlike reports of adult mice and despite massive induction under DFP of c-Fos70 years). Therefore, the extreme retinal sensitivity to AChE modulation may reflect non-catalytic function(s) of AChE in adult photoreceptors. These findings exclude AChE as causing the rd phenotype, suggest that its primary function(s) in mammalian retinal development are non-catalytic ones and indicate special role(s) for the AChE protein in adult photoreceptors.

Cholinotoxic effects on acetylcholinesterase gene expression are associated with brain-region specific alterations in G,C-rich transcripts

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

Entry of Herpes Simplex Virus Type 1 into Cells — Early Steps in Virus Pathogenicity

The entry of herpes simplex virus type 1 (HSV-1) virions into cells represents the initial stage in the process by which HSV-1 introduces its genetic information into a living cell. The molecular basis of virus entry into cells is the ability of the virion’s structural proteins to recognize and interact with protein components of the cell membrane serving as virus receptors. Electron microscopic studies of the cell-associated virions revealed that many virions bind to the cell membrane, but some virions bind to pits in the cell membrane which are either uncoated or clathrincoated pits (Rosenthal et al. 1984). The virions that bind to the cell membrane enter the cell cytoplasm by a fusion mechanism, while the virions attached to the membrane pits enter the cell cytoplasm by the endocytosis mechanism.

Antisense Oligodeoxynucleotide Dependent Suppression of Acetylcholinesterase Expression Reduces Process Extension from Primary Mammalian Neurons

The only currently approved drugs for Alzheimer’s disease (AD) are potent blockers of acetylcholinesterase (AChE) activity (Knapp et al., 1994). However, several lines of evidence suggest novel, non-catalytic morphogenic properties of AChE in process extension (Small et al, 1995; Layer and Willbold, 1995; Jones et al., 1995; Darboux et al., 1996; Sternfeld et al., 1997) and amyloid fibril formation (Inestrosa et al, 1996). This calls for the development of alternative approaches in which both AChE protein synthesis and enzymatic activity would be suppressed, such as the “antisense”.technology (Grifman et al., 1997). To this end, we have designed seven synthetic 3′-phosphorothioatedoligonucleotides (AS-ODNs) targeted towards AChEmRNA and tested their AChE suppression efficacies on the rat neuroendocrine pheochromocytoma cell line, PC 12. Two of these AS-ODNs suppressed the catalytic activity of AChE in nerve growth factor (NGF) -treated PC 12 cells by 25–35%, significantly more than the parallel suppression by control ODNs (Grifman and Soreq, 1997). To study the involvement of AChE in neurite outgrowth and differentiation of primary neurons, we added these two AS-ODNs to primary neuronal cultures from embryonic (El4) mouse whole brain.

Computer Analysis of the Protein Coded by Herpes Simplex Virus Type 1 UL56 Gene

The UL56 gene of herpes simplex virus (HSV) Was studied with the aid of computer programs. These programs analyze the protein amino acid sequence and determine the hydrophilicity, surface probability, flexibility, and antigenicity index. We were able to recognize three different putative domains within this protein which may suggest a role for the UL56 gene product in HSV-1 intraperitoneal infection in mice.

Genetic manipulations of cholinergic communication reveal trans-acting control mechanisms over acetylcholine receptors.

Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate alpha-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the alpha 7 neuronal nicotinic acetylcholine receptor in PC12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of alpha 7 receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.