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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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Related in: MedlinePlus

Loss of PKA-C3 causes behavioral and degenerative phenotypes.A. Inducing a UAS-PKA-C3RNAi construct pan-neuronally with Appl-GAL4 (Appl>PKA-C3RNAi) did not result in a significant reduction in performance compared to controls (Appl>lacZ) when only one copy of each construct is used (het.). However, when using two copies of the RNAi construct (hom.), the flies performed significantly worse than controls. All flies were 14 d old females. n = is number of groups (10–15 flies) tested. Analysis was done using one-way ANOVA with a Dunett's post test and the variance was not different. **p<0.01. B. Measuring the vacuole area in 14 d old Appl>dcr;PKA-C3RNAi flies did not reveal a significant difference to age-matched Appl>dcr controls. However, when aging the PKA-C3 knock-down flies for 30 d they showed significantly increased vacuolization compared to controls. n = number of analyzed flies. Student's t-test were used to compare vacuole size between flies of a given age. The variance was not significantly different when comparing 14 d old flies but were different when comparing 30 d old flies; p<0.001. *p<0.05. C. Model showing the interactions between SWS (light grey) and PKA-C3 (dark grey) and the proposed effects of TOCP. SWS binds to the PKA-C3 subunit via its interaction domain which contains the conserved arginine (R) that is required for binding and that is mutated in SWSR133A. S indicates the serine to which organophosphates bind. Canonical regulatory PKA subunits form dimers and release the catalytic subunits upon binding of cAMP. Although this has not been confirmed for SWS yet, potential cyclic nucleotide binding sites have been identified in SWS. In our model, binding of TOCP (or its metabolite SCOP) and the following aging reaction causes a conformational change that prevents the release and activation of PKA-C3, possibly by interfering with cyclic nucleotide binding.
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pone-0087526-g011: Loss of PKA-C3 causes behavioral and degenerative phenotypes.A. Inducing a UAS-PKA-C3RNAi construct pan-neuronally with Appl-GAL4 (Appl>PKA-C3RNAi) did not result in a significant reduction in performance compared to controls (Appl>lacZ) when only one copy of each construct is used (het.). However, when using two copies of the RNAi construct (hom.), the flies performed significantly worse than controls. All flies were 14 d old females. n = is number of groups (10–15 flies) tested. Analysis was done using one-way ANOVA with a Dunett's post test and the variance was not different. **p<0.01. B. Measuring the vacuole area in 14 d old Appl>dcr;PKA-C3RNAi flies did not reveal a significant difference to age-matched Appl>dcr controls. However, when aging the PKA-C3 knock-down flies for 30 d they showed significantly increased vacuolization compared to controls. n = number of analyzed flies. Student's t-test were used to compare vacuole size between flies of a given age. The variance was not significantly different when comparing 14 d old flies but were different when comparing 30 d old flies; p<0.001. *p<0.05. C. Model showing the interactions between SWS (light grey) and PKA-C3 (dark grey) and the proposed effects of TOCP. SWS binds to the PKA-C3 subunit via its interaction domain which contains the conserved arginine (R) that is required for binding and that is mutated in SWSR133A. S indicates the serine to which organophosphates bind. Canonical regulatory PKA subunits form dimers and release the catalytic subunits upon binding of cAMP. Although this has not been confirmed for SWS yet, potential cyclic nucleotide binding sites have been identified in SWS. In our model, binding of TOCP (or its metabolite SCOP) and the following aging reaction causes a conformational change that prevents the release and activation of PKA-C3, possibly by interfering with cyclic nucleotide binding.

Mentions: If the decrease in PKA-C3 activity is causing TOCP-induced phenotypes, reducing PKA-C3 levels by genetic means should have a similar effect. To test this, we used an RNAi construct against PKA-C3 and expressed it pan-neuronally using Appl-GAL4. Performing fast phototaxis assays in 14 d old flies carrying on copy of the driver and one copy of the RNAi construct (Appl>PKA-C3RNAi het.) did not reveal a significant difference to the control flies expressing lacZ (Fig. 11A). However, when we used flies with two copies of the RNAi construct (hom.), they performed significantly worse (49±3.4% versus controls with 69±5.8%). Performing qPCRs, we found a 22% decrease in PKA-C3 mRNA levels but due to the large variation and the resulting SEM of 31% this was not significant. Heterozygous knockdown flies showed a decrease of 38±12% and this was statistically significant to the controls (Fig. S8). Finally, we investigated whether PKA-C3 knock-down flies show vacuole formation. To increase the effectiveness of the knock-down, we also expressed dicer in these flies. Although 14 d old Appl-GAL4;UAS-PKA-C3RNAi;UAS-dcr flies showed more vacuolization the difference was not significantly different from Appl-GAL4;UAS-dcr control flies (44.7±8.7 µm2 versus 27.9±6.3 µm2). However, when aged for 30 d the knock-down showed more degeneration with a vacuole area of 192.1±32.5 µm2 compared to 63±11.4 µm2 in the controls (Fig. 11B; p = 0.023).


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)

Loss of PKA-C3 causes behavioral and degenerative phenotypes.A. Inducing a UAS-PKA-C3RNAi construct pan-neuronally with Appl-GAL4 (Appl>PKA-C3RNAi) did not result in a significant reduction in performance compared to controls (Appl>lacZ) when only one copy of each construct is used (het.). However, when using two copies of the RNAi construct (hom.), the flies performed significantly worse than controls. All flies were 14 d old females. n = is number of groups (10–15 flies) tested. Analysis was done using one-way ANOVA with a Dunett's post test and the variance was not different. **p<0.01. B. Measuring the vacuole area in 14 d old Appl>dcr;PKA-C3RNAi flies did not reveal a significant difference to age-matched Appl>dcr controls. However, when aging the PKA-C3 knock-down flies for 30 d they showed significantly increased vacuolization compared to controls. n = number of analyzed flies. Student's t-test were used to compare vacuole size between flies of a given age. The variance was not significantly different when comparing 14 d old flies but were different when comparing 30 d old flies; p<0.001. *p<0.05. C. Model showing the interactions between SWS (light grey) and PKA-C3 (dark grey) and the proposed effects of TOCP. SWS binds to the PKA-C3 subunit via its interaction domain which contains the conserved arginine (R) that is required for binding and that is mutated in SWSR133A. S indicates the serine to which organophosphates bind. Canonical regulatory PKA subunits form dimers and release the catalytic subunits upon binding of cAMP. Although this has not been confirmed for SWS yet, potential cyclic nucleotide binding sites have been identified in SWS. In our model, binding of TOCP (or its metabolite SCOP) and the following aging reaction causes a conformational change that prevents the release and activation of PKA-C3, possibly by interfering with cyclic nucleotide binding.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087526-g011: Loss of PKA-C3 causes behavioral and degenerative phenotypes.A. Inducing a UAS-PKA-C3RNAi construct pan-neuronally with Appl-GAL4 (Appl>PKA-C3RNAi) did not result in a significant reduction in performance compared to controls (Appl>lacZ) when only one copy of each construct is used (het.). However, when using two copies of the RNAi construct (hom.), the flies performed significantly worse than controls. All flies were 14 d old females. n = is number of groups (10–15 flies) tested. Analysis was done using one-way ANOVA with a Dunett's post test and the variance was not different. **p<0.01. B. Measuring the vacuole area in 14 d old Appl>dcr;PKA-C3RNAi flies did not reveal a significant difference to age-matched Appl>dcr controls. However, when aging the PKA-C3 knock-down flies for 30 d they showed significantly increased vacuolization compared to controls. n = number of analyzed flies. Student's t-test were used to compare vacuole size between flies of a given age. The variance was not significantly different when comparing 14 d old flies but were different when comparing 30 d old flies; p<0.001. *p<0.05. C. Model showing the interactions between SWS (light grey) and PKA-C3 (dark grey) and the proposed effects of TOCP. SWS binds to the PKA-C3 subunit via its interaction domain which contains the conserved arginine (R) that is required for binding and that is mutated in SWSR133A. S indicates the serine to which organophosphates bind. Canonical regulatory PKA subunits form dimers and release the catalytic subunits upon binding of cAMP. Although this has not been confirmed for SWS yet, potential cyclic nucleotide binding sites have been identified in SWS. In our model, binding of TOCP (or its metabolite SCOP) and the following aging reaction causes a conformational change that prevents the release and activation of PKA-C3, possibly by interfering with cyclic nucleotide binding.
Mentions: If the decrease in PKA-C3 activity is causing TOCP-induced phenotypes, reducing PKA-C3 levels by genetic means should have a similar effect. To test this, we used an RNAi construct against PKA-C3 and expressed it pan-neuronally using Appl-GAL4. Performing fast phototaxis assays in 14 d old flies carrying on copy of the driver and one copy of the RNAi construct (Appl>PKA-C3RNAi het.) did not reveal a significant difference to the control flies expressing lacZ (Fig. 11A). However, when we used flies with two copies of the RNAi construct (hom.), they performed significantly worse (49±3.4% versus controls with 69±5.8%). Performing qPCRs, we found a 22% decrease in PKA-C3 mRNA levels but due to the large variation and the resulting SEM of 31% this was not significant. Heterozygous knockdown flies showed a decrease of 38±12% and this was statistically significant to the controls (Fig. S8). Finally, we investigated whether PKA-C3 knock-down flies show vacuole formation. To increase the effectiveness of the knock-down, we also expressed dicer in these flies. Although 14 d old Appl-GAL4;UAS-PKA-C3RNAi;UAS-dcr flies showed more vacuolization the difference was not significantly different from Appl-GAL4;UAS-dcr control flies (44.7±8.7 µm2 versus 27.9±6.3 µm2). However, when aged for 30 d the knock-down showed more degeneration with a vacuole area of 192.1±32.5 µm2 compared to 63±11.4 µm2 in the controls (Fig. 11B; p = 0.023).

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