The primary objective of this proposal is to investigate the underlying mechanisms of organophosphorus compound-induced delayed neurotoxicity (OPIDN). OPIDN is a distal axonopathy of the peripheral and central nervous systems and is characterized by axonal swellings containing aggregates of cytoskeletal proteins i.e., neurofilaments and tubulin. Within the past decade we have demonstrated that pathognomonic of OPIDN is an aberrant increased autophosphorylation of calcium/calmodulin kinase II (CaM Kinase II) and increased phosphorylation of cytoskeletal proteins i.e., MAP 2, tubulin, neurofilament triplet proteins, and myelin basic protein. Concurrent with increased phosphorylation is an enhancement in calmodulin binding to CaM Kinase II. In this proposal we specifically propose to 1) study the regulation of CaM Kinase II autophosphorylation and the phosphorylation of its substrate cytoskeletal proteins; 2) characterize the protein makeup of the axonal swellings; 3) correlate the phosphorylation of cytoskeletal proteins with axonal degeneration. To determine the regulatory mechanisms involved in protein phosphorylation, the in vivo phosphorylation state of CaM Kinase II and its various substrate cytoskeletal proteins in 0P treated and control hens will be determined. To determine the extent of isoforms of CaM Kinase present and their involvement in the development of OPIDN, we plan to construct a cDNA library to the hen enzyme to probe for alterations in the alpha and beta mRNA's upon 0P treatment. Secondly, we will determine the nature of the cytoskeletal aggregates with respect to their phosphorylation state and time and spatial distribution. This will be accomplished by implementing immunohistochemical techniques. Lastly, we will test the hypothesis that aberrant increased phosphorylation of neurofilaments decreases their axonal transport rate which results in their accumulation at distal parts of the axons. We will determine the rates of axonal transport of specific cytoskeletal proteins in the presence or absence of OPs. [3H]Proline and [32p]orthophosphate will be used to radiolabel the cytoskeletal proteins and visualize them electrophoretically following segmental analysis. Cumulatively, our goal is to demonstrate that functional changes in CaM Kinase II lead to increased phosphorylation and aggregation of cytoskeletal elements resulting in axonal degeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES005154-09
Application #
3253387
Study Section
Safety and Occupational Health Study Section (SOH)
Project Start
1988-09-01
Project End
1996-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Damodaran, T V; Attia, M K; Abou-Donia, M B (2011) Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: a study of molecular, cellular, and anatomical parameters. Toxicol Appl Pharmacol 256:348-59
Damodaran, Tirupapuliyur V; Gupta, Ram P; Attia, Moustafa K et al. (2009) DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of beta-tubulin subtypes in the CNS of hens contributes to OPIDN. Toxicol Appl Pharmacol 240:132-42
Damodaran, T V; Rahman, A A; Abou-Donia, M B (2000) Early differential induction of C-jun in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP). Neurochem Res 25:1579-86
Gupta, R P; Abou-Donia, M B (1999) Tau phosphorylation by diisopropyl phosphorofluoridate (DFP)-treated hen brain supernatant inhibits its binding with microtubules: role of Ca2+/Calmodulin-dependent protein kinase II in tau phosphorylation. Arch Biochem Biophys 365:268-78
Gupta, R P; Abou-Donia, M B (1998) Tau proteins-enhanced Ca2+/calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II. Brain Res 813:32-43
Knoth-Anderson, J; Abou-Donia, M B (1993) Differential effects of triphenylphosphite and diisopropyl phosphorofluoridate on catecholamine secretion from bovine adrenomedullary chromaffin cells. J Toxicol Environ Health 38:103-14
Abou-Donia, M B; Viana, M E; Gupta, R P et al. (1993) Enhanced calmodulin binding concurrent with increased kinase-dependent phosphorylation of cytoskeletal proteins following a single subcutaneous injection of diisopropyl phosphorofluoridate in hens. Neurochem Int 22:165-73
Gupta, R P; Lapadula, D M; Abou-Donia, M B (1992) Ca2+/calmodulin-dependent protein kinase II from hen brain. Purification and characterization. Biochem Pharmacol 43:1975-88
Jensen, K F; Lapadula, D M; Anderson, J K et al. (1992) Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with tri-ortho-cresyl phosphate (TOCP). J Neurosci Res 33:455-60
Lapadula, E S; Lapadula, D M; Abou-Donia, M B (1992) Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins. Neurochem Int 20:247-55

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