Nerve agent exposure elicits site-specific changes in protein phosphorylation in mouse brain
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Published on: February 4, 2014
Hongwen Zhu1, Jennifer J O'Brien, James P O'Callaghan
1Department of Molecular Neuropharmacology, Intra-Cellular Therapies, Inc. (ITI), Audubon Business and Technology Center, 3960 Broadway, New York, NY 10032, USA.
Abstract:
Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies.
Insights
Organophosphorus compounds cause severe toxicity by inhibiting acetylcholinesterase (AChE). This study reveals region-specific brain signaling changes after OP exposure, identifying potential targets for new anti-convulsant therapies.
Area of Science:
- Neuroscience
- Toxicology
- Biochemistry
Background:
- Organophosphorus (OP) compounds irreversibly inhibit acetylcholinesterase (AChE), leading to severe neurotoxicity, including seizures, coma, and death.
- Understanding the central nervous system (CNS) pathways affected by OP intoxication is crucial for developing effective treatments against acute toxicity and long-term cognitive deficits.
Purpose of the Study:
- To investigate the changes in neuronal phosphoprotein phosphorylation in response to OP exposure using the CNSProfile method.
- To identify specific CNS pathways and molecular targets affected by OP compounds, such as diisopropyl fluorophosphate (DFP).
Main Methods:
- Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in the brains of DFP-treated mice.
- Hippocampus and striatum tissues were analyzed by immunoblotting with phospho-specific antibodies to detect changes in protein phosphorylation.
- The effects of phencynonate hydrochloride, a muscarinic cholinergic antagonist, on DFP-induced behaviors and phosphorylation changes were evaluated.
Main Results:
- DFP exposure altered the phosphorylation of key neuronal proteins, including NR1 NMDA receptors and DARPP-32, in a region-specific manner (striatum vs. hippocampus).
- NR1 phosphorylation was decreased in the striatum but increased in the hippocampus following DFP treatment.
- DARPP-32 phosphorylation at Threonine75 (T75), a Cdk5 kinase substrate, was selectively increased in the striatum.
- Phencynonate hydrochloride administration prevented DFP-induced seizure-like behaviors and normalized the observed phosphorylation changes.
Conclusions:
- OP nerve agent exposure induces region-specific alterations in intracellular signaling pathways within the brain.
- These signaling changes correlate with seizure-like behavior and are reversible with muscarinic receptor blockade.
- The study identifies specific targets, including Cdk5 kinase substrates, that may serve as a basis for novel anti-convulsant therapies against OP intoxication.
