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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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TOCP treatment reduces PKA activity.A. Flies treated with 16 mg/ml TOCP reveal a significant reduction in PKA activity in head extracts whereas flies treated with Paraoxon (PO; 0.2 mg/ml) show no change in PKA activity. B. Mouse NTE binds fly PKA-C3 in Two-Hybrid assays, whereas it does not interact with the other two known fly catalytic subunits, PKA-C2 and PKA-C1. C. TOCP treatment (14 µg/ml) reduces PKA activity in cultured rat hippocampal neurons whereas exposure to Paraoxon (PO; 4 µg/ml) does not. n = is number of independent PKA assays. PKA activity is given as the ratio of the luminosity value of phosphorylated to unphosphorylated kemptide peptide per µg protein. Student's t-tests were used to compare treated and untreated flies or hippocampal neurons. SEMs are indicated. *p<0.05, **p<0.01. (The variances are not significantly different).
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pone-0087526-g008: TOCP treatment reduces PKA activity.A. Flies treated with 16 mg/ml TOCP reveal a significant reduction in PKA activity in head extracts whereas flies treated with Paraoxon (PO; 0.2 mg/ml) show no change in PKA activity. B. Mouse NTE binds fly PKA-C3 in Two-Hybrid assays, whereas it does not interact with the other two known fly catalytic subunits, PKA-C2 and PKA-C1. C. TOCP treatment (14 µg/ml) reduces PKA activity in cultured rat hippocampal neurons whereas exposure to Paraoxon (PO; 4 µg/ml) does not. n = is number of independent PKA assays. PKA activity is given as the ratio of the luminosity value of phosphorylated to unphosphorylated kemptide peptide per µg protein. Student's t-tests were used to compare treated and untreated flies or hippocampal neurons. SEMs are indicated. *p<0.05, **p<0.01. (The variances are not significantly different).

Mentions: We previously showed that SWS, besides acting as a phospholipase, also binds the C3 catalytic subunit of PKA, thereby inhibiting PKA activity [24]. We therefore tested whether the PKA function is affected by TOCP treatment. Measuring PKA activity in head extracts from flies fed with 16 mg/ml TOCP for 24 h resulted in a significant decrease in PKA activity when compared to vehicle treated flies (Fig. 8A), suggesting that TOCP interferes with the release and activation of PKA-C3. In contrast, treating flies with the non-neuropathic paraoxon (0.2 mg/ml, the flies did not survive higher doses) did not result in decreased PKA activity in two independent measurements (Fig. 8A). To determine whether this effect could also play a role in the human syndrome and because the PKA function had not been confirmed for vertebrate NTE, we performed Two-Hybrid assays with mouse NTE (mNTE). Using the three catalytic subunits known in Drosophila showed that mNTE binds PKA-C3, but not the other two catalytic subunits (Fig. 8B). This shows that vertebrate NTE, like SWS [24], specifically binds to the C3 subunit, further confirming the functional conservation of SWS and NTE. After verifying that NTE can interact with the PKA-C3 catalytic subunit, we treated cultured hippocampal neurons derived from rats with TOCP and measured the effect on PKA activity (rat NTE is 99% identical to mouse NTE and 95% identical to human NTE). Treating these neurons with a dose of 14 µg/ml TOCP for 1 d also resulted in a significant reduction in PKA activity (Fig. 8C) suggesting that TOCP-induced changes in the activity of the vertebrate orthologues of PKA-C3, Pkare in mouse and PrKX in humans, is part of the toxic mechanism of TOCP (the mouse and the rat protein are 95% identical). Again, treatment with paraoxon at 4 µg/ml had no effect on PKA activity. The effect of TOCP exposure on PKA activity was not due to an increased lethality of cells because treatment resulted in 9.44±0.32% dead cells versus 7.9±2.7% in mock treated cells (p = 0.74). Neither was this effect due to an effect on PKA-C3 levels because treatment with TOCP did not change the levels of PKA-C3 nor SWS (Fig. S7).


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)

TOCP treatment reduces PKA activity.A. Flies treated with 16 mg/ml TOCP reveal a significant reduction in PKA activity in head extracts whereas flies treated with Paraoxon (PO; 0.2 mg/ml) show no change in PKA activity. B. Mouse NTE binds fly PKA-C3 in Two-Hybrid assays, whereas it does not interact with the other two known fly catalytic subunits, PKA-C2 and PKA-C1. C. TOCP treatment (14 µg/ml) reduces PKA activity in cultured rat hippocampal neurons whereas exposure to Paraoxon (PO; 4 µg/ml) does not. n = is number of independent PKA assays. PKA activity is given as the ratio of the luminosity value of phosphorylated to unphosphorylated kemptide peptide per µg protein. Student's t-tests were used to compare treated and untreated flies or hippocampal neurons. SEMs are indicated. *p<0.05, **p<0.01. (The variances are not significantly different).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928115&req=5

pone-0087526-g008: TOCP treatment reduces PKA activity.A. Flies treated with 16 mg/ml TOCP reveal a significant reduction in PKA activity in head extracts whereas flies treated with Paraoxon (PO; 0.2 mg/ml) show no change in PKA activity. B. Mouse NTE binds fly PKA-C3 in Two-Hybrid assays, whereas it does not interact with the other two known fly catalytic subunits, PKA-C2 and PKA-C1. C. TOCP treatment (14 µg/ml) reduces PKA activity in cultured rat hippocampal neurons whereas exposure to Paraoxon (PO; 4 µg/ml) does not. n = is number of independent PKA assays. PKA activity is given as the ratio of the luminosity value of phosphorylated to unphosphorylated kemptide peptide per µg protein. Student's t-tests were used to compare treated and untreated flies or hippocampal neurons. SEMs are indicated. *p<0.05, **p<0.01. (The variances are not significantly different).
Mentions: We previously showed that SWS, besides acting as a phospholipase, also binds the C3 catalytic subunit of PKA, thereby inhibiting PKA activity [24]. We therefore tested whether the PKA function is affected by TOCP treatment. Measuring PKA activity in head extracts from flies fed with 16 mg/ml TOCP for 24 h resulted in a significant decrease in PKA activity when compared to vehicle treated flies (Fig. 8A), suggesting that TOCP interferes with the release and activation of PKA-C3. In contrast, treating flies with the non-neuropathic paraoxon (0.2 mg/ml, the flies did not survive higher doses) did not result in decreased PKA activity in two independent measurements (Fig. 8A). To determine whether this effect could also play a role in the human syndrome and because the PKA function had not been confirmed for vertebrate NTE, we performed Two-Hybrid assays with mouse NTE (mNTE). Using the three catalytic subunits known in Drosophila showed that mNTE binds PKA-C3, but not the other two catalytic subunits (Fig. 8B). This shows that vertebrate NTE, like SWS [24], specifically binds to the C3 subunit, further confirming the functional conservation of SWS and NTE. After verifying that NTE can interact with the PKA-C3 catalytic subunit, we treated cultured hippocampal neurons derived from rats with TOCP and measured the effect on PKA activity (rat NTE is 99% identical to mouse NTE and 95% identical to human NTE). Treating these neurons with a dose of 14 µg/ml TOCP for 1 d also resulted in a significant reduction in PKA activity (Fig. 8C) suggesting that TOCP-induced changes in the activity of the vertebrate orthologues of PKA-C3, Pkare in mouse and PrKX in humans, is part of the toxic mechanism of TOCP (the mouse and the rat protein are 95% identical). Again, treatment with paraoxon at 4 µg/ml had no effect on PKA activity. The effect of TOCP exposure on PKA activity was not due to an increased lethality of cells because treatment resulted in 9.44±0.32% dead cells versus 7.9±2.7% in mock treated cells (p = 0.74). Neither was this effect due to an effect on PKA-C3 levels because treatment with TOCP did not change the levels of PKA-C3 nor SWS (Fig. S7).

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