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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 induces lethality.A. Wild type fly fed on glucose. B. A wild type fly fed with glucose containing TOCP and blue food coloring shows food uptake by the blue coloring of the abdomen and proboscis (arrows). C. Survival of flies treated with different concentrations of TOCP. n =  number of independent tests with 10–15 flies. Analysis was done using one-way ANOVA with a Dunett's post test to compare the treated flies to mock treated flies. The SEMs are indicated. *p<0.05, **p<0.0. (the variance was not significantly different with the exception of day 14 with p = 0.02)
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pone-0087526-g001: TOCP treatment induces lethality.A. Wild type fly fed on glucose. B. A wild type fly fed with glucose containing TOCP and blue food coloring shows food uptake by the blue coloring of the abdomen and proboscis (arrows). C. Survival of flies treated with different concentrations of TOCP. n =  number of independent tests with 10–15 flies. Analysis was done using one-way ANOVA with a Dunett's post test to compare the treated flies to mock treated flies. The SEMs are indicated. *p<0.05, **p<0.0. (the variance was not significantly different with the exception of day 14 with p = 0.02)

Mentions: To establish Drosophila as a model for OP-induced neurotoxicity, flies were treated with the organophosphorous compound tri-ortho cresyl phosphate (TOCP). We chose TOCP because, in contrast to most other organophosphates, TOCP appears to have minimal or no effects on acetylcholine esterase (AChE) activity [18]. In addition, TOCP is the compound that originally lead to the identification of OPIDN (Smith, 1930) and it has been used to experimentally induce OPIDN in chickens [17]. We first determined an appropriate dose by treating flies with increasing doses of TOCP, ranging from 0.08 mg to 32 mg per ml glucose solution, which is approximately 0.5 to 150 times the neuropathic oral dose for humans with a fly weighing approximately 1 mg and drinking about 1 µl in the 1 d treatment period [32]. To confirm that the flies consumed the TOCP containing glucose solution, we added food color which was easily detectable after feeding (Fig. 1B, untreated fly in Fig. 1A), confirming that the flies did take up the food. Whereas the lowest concentrations of 0.08 mg/ml did not result in an increased lethality compared to mock treated flies (Fig. 1C), intermediated doses of 8 mg/ml and 16 mg/ml caused a significant increase in lethality after 14 d. These doses did not induce significant lethality after 2 d, showing that they had a normal survival rate during the treatment period. In contrast, the highest dose of 32 mg/ml resulted in the death of 40% of the flies already two days after treatment and 50% after two weeks (Fig. 1C). Although we cannot distinguish whether the lethality is solely due to neuropathologic effects or to effects of TOCP on other tissues, these results show that TOCP treatment is deleterious to flies.


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 induces lethality.A. Wild type fly fed on glucose. B. A wild type fly fed with glucose containing TOCP and blue food coloring shows food uptake by the blue coloring of the abdomen and proboscis (arrows). C. Survival of flies treated with different concentrations of TOCP. n =  number of independent tests with 10–15 flies. Analysis was done using one-way ANOVA with a Dunett's post test to compare the treated flies to mock treated flies. The SEMs are indicated. *p<0.05, **p<0.0. (the variance was not significantly different with the exception of day 14 with p = 0.02)
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087526-g001: TOCP treatment induces lethality.A. Wild type fly fed on glucose. B. A wild type fly fed with glucose containing TOCP and blue food coloring shows food uptake by the blue coloring of the abdomen and proboscis (arrows). C. Survival of flies treated with different concentrations of TOCP. n =  number of independent tests with 10–15 flies. Analysis was done using one-way ANOVA with a Dunett's post test to compare the treated flies to mock treated flies. The SEMs are indicated. *p<0.05, **p<0.0. (the variance was not significantly different with the exception of day 14 with p = 0.02)
Mentions: To establish Drosophila as a model for OP-induced neurotoxicity, flies were treated with the organophosphorous compound tri-ortho cresyl phosphate (TOCP). We chose TOCP because, in contrast to most other organophosphates, TOCP appears to have minimal or no effects on acetylcholine esterase (AChE) activity [18]. In addition, TOCP is the compound that originally lead to the identification of OPIDN (Smith, 1930) and it has been used to experimentally induce OPIDN in chickens [17]. We first determined an appropriate dose by treating flies with increasing doses of TOCP, ranging from 0.08 mg to 32 mg per ml glucose solution, which is approximately 0.5 to 150 times the neuropathic oral dose for humans with a fly weighing approximately 1 mg and drinking about 1 µl in the 1 d treatment period [32]. To confirm that the flies consumed the TOCP containing glucose solution, we added food color which was easily detectable after feeding (Fig. 1B, untreated fly in Fig. 1A), confirming that the flies did take up the food. Whereas the lowest concentrations of 0.08 mg/ml did not result in an increased lethality compared to mock treated flies (Fig. 1C), intermediated doses of 8 mg/ml and 16 mg/ml caused a significant increase in lethality after 14 d. These doses did not induce significant lethality after 2 d, showing that they had a normal survival rate during the treatment period. In contrast, the highest dose of 32 mg/ml resulted in the death of 40% of the flies already two days after treatment and 50% after two weeks (Fig. 1C). Although we cannot distinguish whether the lethality is solely due to neuropathologic effects or to effects of TOCP on other tissues, these results show that TOCP treatment is deleterious to flies.

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