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Organophosphate-induced changes in the PKA regulatory function of Swiss Cheese/NTE lead to behavioral deficits and neurodegeneration.

Wentzell JS, Cassar M, Kretzschmar D - PLoS ONE (2014)

Bottom Line: Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models.In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture.Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Occupational and Environmental Toxicology, Oregon Health & Sciences University, Portland, Oregon, United States of America.

ABSTRACT
Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity.

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PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.A. Flies expressing additional PKA-C3 in neurons via elav-GAL4 do not show the TOCP-induced reduction in performance seen in elav>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWSR133A construct are protected against TOCP-induced behavioral deficits. In addition, these flies do not show the reduction in performance observed in untreated flies overexpressing the wild type SWS construct (elav>SWS). B. Although PKA-C3 overexpressing flies show a significant increase in vacuole formation when untreated, TOCP treatment does not enhance this phenotype, but significantly reduces vacuole formation. C. PKA-C3 overexpression has no effect on the neurite shortening observed after TOCP treatment of primary neurons. n = is number of groups tested with 10–20 female flies each in A, n = number of cells or head sections analyzed in B and C. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in B. All flies used in the fast phototaxis assays were 14 d old females. SEMs are indicated in all graphs. *p<0.05, **p<0.01, ***p<0.001. (The variances were not significantly different in the tests done to compare vacuole size and behavioral deficits, but were different between treated and untreated cells: p<0.001).
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pone-0087526-g009: PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.A. Flies expressing additional PKA-C3 in neurons via elav-GAL4 do not show the TOCP-induced reduction in performance seen in elav>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWSR133A construct are protected against TOCP-induced behavioral deficits. In addition, these flies do not show the reduction in performance observed in untreated flies overexpressing the wild type SWS construct (elav>SWS). B. Although PKA-C3 overexpressing flies show a significant increase in vacuole formation when untreated, TOCP treatment does not enhance this phenotype, but significantly reduces vacuole formation. C. PKA-C3 overexpression has no effect on the neurite shortening observed after TOCP treatment of primary neurons. n = is number of groups tested with 10–20 female flies each in A, n = number of cells or head sections analyzed in B and C. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in B. All flies used in the fast phototaxis assays were 14 d old females. SEMs are indicated in all graphs. *p<0.05, **p<0.01, ***p<0.001. (The variances were not significantly different in the tests done to compare vacuole size and behavioral deficits, but were different between treated and untreated cells: p<0.001).

Mentions: The results described above suggest that a decrease in PKA-C3 activity plays a role in the behavioral and degenerative defects occurring after TOCP exposure. To further support this, we treated flies that expressed additional PKA-C3 in neurons via elav-GAL4 with 8 mg/ml TOCP as described above and performed fast phototaxis assays. As shown in figure 9A, these flies were protected from behavioral deficits with a performance index of 71±4.1%, which is not different from the value of untreated PKA-C3 overexpressing flies (69±3.0%) or untreated control flies (elav>lacZ; 68±2.2%).


Organophosphate-induced changes in the PKA regulatory function of Swiss Cheese/NTE lead to behavioral deficits and neurodegeneration.

Wentzell JS, Cassar M, Kretzschmar D - PLoS ONE (2014)

PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.A. Flies expressing additional PKA-C3 in neurons via elav-GAL4 do not show the TOCP-induced reduction in performance seen in elav>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWSR133A construct are protected against TOCP-induced behavioral deficits. In addition, these flies do not show the reduction in performance observed in untreated flies overexpressing the wild type SWS construct (elav>SWS). B. Although PKA-C3 overexpressing flies show a significant increase in vacuole formation when untreated, TOCP treatment does not enhance this phenotype, but significantly reduces vacuole formation. C. PKA-C3 overexpression has no effect on the neurite shortening observed after TOCP treatment of primary neurons. n = is number of groups tested with 10–20 female flies each in A, n = number of cells or head sections analyzed in B and C. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in B. All flies used in the fast phototaxis assays were 14 d old females. SEMs are indicated in all graphs. *p<0.05, **p<0.01, ***p<0.001. (The variances were not significantly different in the tests done to compare vacuole size and behavioral deficits, but were different between treated and untreated cells: p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928115&req=5

pone-0087526-g009: PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.A. Flies expressing additional PKA-C3 in neurons via elav-GAL4 do not show the TOCP-induced reduction in performance seen in elav>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWSR133A construct are protected against TOCP-induced behavioral deficits. In addition, these flies do not show the reduction in performance observed in untreated flies overexpressing the wild type SWS construct (elav>SWS). B. Although PKA-C3 overexpressing flies show a significant increase in vacuole formation when untreated, TOCP treatment does not enhance this phenotype, but significantly reduces vacuole formation. C. PKA-C3 overexpression has no effect on the neurite shortening observed after TOCP treatment of primary neurons. n = is number of groups tested with 10–20 female flies each in A, n = number of cells or head sections analyzed in B and C. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in B. All flies used in the fast phototaxis assays were 14 d old females. SEMs are indicated in all graphs. *p<0.05, **p<0.01, ***p<0.001. (The variances were not significantly different in the tests done to compare vacuole size and behavioral deficits, but were different between treated and untreated cells: p<0.001).
Mentions: The results described above suggest that a decrease in PKA-C3 activity plays a role in the behavioral and degenerative defects occurring after TOCP exposure. To further support this, we treated flies that expressed additional PKA-C3 in neurons via elav-GAL4 with 8 mg/ml TOCP as described above and performed fast phototaxis assays. As shown in figure 9A, these flies were protected from behavioral deficits with a performance index of 71±4.1%, which is not different from the value of untreated PKA-C3 overexpressing flies (69±3.0%) or untreated control flies (elav>lacZ; 68±2.2%).

Bottom Line: Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models.In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture.Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Occupational and Environmental Toxicology, Oregon Health & Sciences University, Portland, Oregon, United States of America.

ABSTRACT
Organophosphate-induced delayed neuropathy (OPIDN) is a Wallerian-type axonopathy that occurs weeks after exposure to certain organophosphates (OPs). OPs have been shown to bind to Neuropathy Target Esterase (NTE), thereby inhibiting its enzymatic activity. However, only OPs that also induce the so-called aging reaction cause OPIDN. This reaction results in the release and possible transfer of a side group from the bound OP to NTE and it has been suggested that this induces an unknown toxic function of NTE. To further investigate the mechanisms of aging OPs, we used Drosophila, which expresses a functionally conserved orthologue of NTE named Swiss Cheese (SWS). Treating flies with the organophosporous compound tri-ortho-cresyl phosphate (TOCP) resulted in behavioral deficits and neurodegeneration two weeks after exposure, symptoms similar to the delayed effects observed in other models. In addition, we found that primary neurons showed signs of axonal degeneration within an hour after treatment. Surprisingly, increasing the levels of SWS, and thereby its enzymatic activity after exposure, did not ameliorate these phenotypes. In contrast, reducing SWS levels protected from TOCP-induced degeneration and behavioral deficits but did not affect the axonopathy observed in cell culture. Besides its enzymatic activity as a phospholipase, SWS also acts as regulatory PKA subunit, binding and inhibiting the C3 catalytic subunit. Measuring PKA activity in TOCP treated flies revealed a significant decrease that was also confirmed in treated rat hippocampal neurons. Flies expressing additional PKA-C3 were protected from the behavioral and degenerative phenotypes caused by TOCP exposure whereas primary neurons were not. In addition, knocking-down PKA-C3 caused similar behavioral and degenerative phenotypes as TOCP treatment. We therefore propose a model in which OP-modified SWS cannot release PKA-C3 and that the resulting loss of PKA-C3 activity plays a crucial role in developing the delayed symptoms of OPIDN but not in the acute toxicity.

Show MeSH
Related in: MedlinePlus