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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 induces neurite shortening in primary neuronal cultures.A. Dose response curve showing that TOCP doses equal or higher than 7 µg/ml cause a significant reduction in neurite length. 30–56 neurons were measured for each condition. B. Live imaging of a neuron treated with 14 µg/ml TOCP reveals the formation of varicosities (arrows) and neurite degeneration (arrowheads) 50 min after the addition of TOCP. Another 50 mins later, these phenotypes are even more pronounced. C. The length of neurites is dramatically reduced in TOCP treated cells versus mock treated cells, but also significantly shorter in TOCP treated cells compared to cells that have been fixed at the time of treatment (left graph). The graph on the right shows the change in length between each condition. Analysis was done using one-way ANOVA with a Dunett's post test to compare to mock treated cells. n = number of cells measured and the SEMs are shown. **p<0.01, ***p<0.001. Scale bar in B = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).
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pone-0087526-g004: TOCP induces neurite shortening in primary neuronal cultures.A. Dose response curve showing that TOCP doses equal or higher than 7 µg/ml cause a significant reduction in neurite length. 30–56 neurons were measured for each condition. B. Live imaging of a neuron treated with 14 µg/ml TOCP reveals the formation of varicosities (arrows) and neurite degeneration (arrowheads) 50 min after the addition of TOCP. Another 50 mins later, these phenotypes are even more pronounced. C. The length of neurites is dramatically reduced in TOCP treated cells versus mock treated cells, but also significantly shorter in TOCP treated cells compared to cells that have been fixed at the time of treatment (left graph). The graph on the right shows the change in length between each condition. Analysis was done using one-way ANOVA with a Dunett's post test to compare to mock treated cells. n = number of cells measured and the SEMs are shown. **p<0.01, ***p<0.001. Scale bar in B = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).

Mentions: Although this revealed that TOCP treatment can lead to neuronal degeneration in flies, this experiment did not allow us to specifically detect axonal degeneration, which is a characteristic sign of OPIDN. We therefore performed primary neuronal cell cultures and treated them after 12 h in culture with different doses of TOCP for 6 h before measuring the longest neurite of each cell. Whereas the two lower doses did not affect the average neurite length when compared to vehicle treated cells, higher doses caused a significant reduction, which correlated with the increasing concentration of TOCP (Fig. 4A). To determine whether the addition of TOCP prevents further outgrowth or leads to retraction/degeneration of neurites, we performed live cell imaging studies. For this and the following studies, we chose the dose of 14 µg/ml which had an intermediate effect with approximately 40% reduction in neurite length in our dosage study. Also this dose did not induce a significant amount of cell death with 8.44±0.32% dead cells in treated neurons (n = 63) versus 7.9±2.7% in untreated ones (n = 58; p = 0.74). For live imaging, cells were allowed to adhere to a coverslip for 12 h, which was then transferred to the microscope chamber and allowed to settle for another 30 min before starting imaging. TOCP was added after 100 min and within 50 minutes of exposure, the neurites started to look thinner and shorter, followed by the formation of varicosities and fragmentation (arrows, Fig. 4B; the arrowheads point to shorter neurites), typical signs of Wallerian degeneration [33]. The percentage of cells with varicosities after TOCP treatment is shown in Fig. S3. In addition, this suggested that TOCP did not just prevent further outgrowth but caused the degeneration of existing neurites. To confirm this result, we fixed one set of neurons after 12 h in culture (pretreat. in Fig. 4C) whereas two other sets were treated with either 14 µg/ml TOCP or the vehicle (veh.). These neurons were then allowed to grow for another 6 h before the average neurite length was determined. As expected, neurite length was significantly shorter in the TOCP treated cells compared to vehicle treated ones, confirming the toxic effects of TOCP (Fig. 4C). But, whereas neurite length was dramatically increased in vehicle treated neurons compared to the ones fixed at treatment time (confirming their continuing growth), the average length of neurites from TOCP treated neurons was significantly shorter compared to the ones fixed 6 h earlier (Fig. 4C). This confirmed that TOCP treatment does result in a retraction/degeneration of established neurites in our model.


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 induces neurite shortening in primary neuronal cultures.A. Dose response curve showing that TOCP doses equal or higher than 7 µg/ml cause a significant reduction in neurite length. 30–56 neurons were measured for each condition. B. Live imaging of a neuron treated with 14 µg/ml TOCP reveals the formation of varicosities (arrows) and neurite degeneration (arrowheads) 50 min after the addition of TOCP. Another 50 mins later, these phenotypes are even more pronounced. C. The length of neurites is dramatically reduced in TOCP treated cells versus mock treated cells, but also significantly shorter in TOCP treated cells compared to cells that have been fixed at the time of treatment (left graph). The graph on the right shows the change in length between each condition. Analysis was done using one-way ANOVA with a Dunett's post test to compare to mock treated cells. n = number of cells measured and the SEMs are shown. **p<0.01, ***p<0.001. Scale bar in B = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087526-g004: TOCP induces neurite shortening in primary neuronal cultures.A. Dose response curve showing that TOCP doses equal or higher than 7 µg/ml cause a significant reduction in neurite length. 30–56 neurons were measured for each condition. B. Live imaging of a neuron treated with 14 µg/ml TOCP reveals the formation of varicosities (arrows) and neurite degeneration (arrowheads) 50 min after the addition of TOCP. Another 50 mins later, these phenotypes are even more pronounced. C. The length of neurites is dramatically reduced in TOCP treated cells versus mock treated cells, but also significantly shorter in TOCP treated cells compared to cells that have been fixed at the time of treatment (left graph). The graph on the right shows the change in length between each condition. Analysis was done using one-way ANOVA with a Dunett's post test to compare to mock treated cells. n = number of cells measured and the SEMs are shown. **p<0.01, ***p<0.001. Scale bar in B = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).
Mentions: Although this revealed that TOCP treatment can lead to neuronal degeneration in flies, this experiment did not allow us to specifically detect axonal degeneration, which is a characteristic sign of OPIDN. We therefore performed primary neuronal cell cultures and treated them after 12 h in culture with different doses of TOCP for 6 h before measuring the longest neurite of each cell. Whereas the two lower doses did not affect the average neurite length when compared to vehicle treated cells, higher doses caused a significant reduction, which correlated with the increasing concentration of TOCP (Fig. 4A). To determine whether the addition of TOCP prevents further outgrowth or leads to retraction/degeneration of neurites, we performed live cell imaging studies. For this and the following studies, we chose the dose of 14 µg/ml which had an intermediate effect with approximately 40% reduction in neurite length in our dosage study. Also this dose did not induce a significant amount of cell death with 8.44±0.32% dead cells in treated neurons (n = 63) versus 7.9±2.7% in untreated ones (n = 58; p = 0.74). For live imaging, cells were allowed to adhere to a coverslip for 12 h, which was then transferred to the microscope chamber and allowed to settle for another 30 min before starting imaging. TOCP was added after 100 min and within 50 minutes of exposure, the neurites started to look thinner and shorter, followed by the formation of varicosities and fragmentation (arrows, Fig. 4B; the arrowheads point to shorter neurites), typical signs of Wallerian degeneration [33]. The percentage of cells with varicosities after TOCP treatment is shown in Fig. S3. In addition, this suggested that TOCP did not just prevent further outgrowth but caused the degeneration of existing neurites. To confirm this result, we fixed one set of neurons after 12 h in culture (pretreat. in Fig. 4C) whereas two other sets were treated with either 14 µg/ml TOCP or the vehicle (veh.). These neurons were then allowed to grow for another 6 h before the average neurite length was determined. As expected, neurite length was significantly shorter in the TOCP treated cells compared to vehicle treated ones, confirming the toxic effects of TOCP (Fig. 4C). But, whereas neurite length was dramatically increased in vehicle treated neurons compared to the ones fixed at treatment time (confirming their continuing growth), the average length of neurites from TOCP treated neurons was significantly shorter compared to the ones fixed 6 h earlier (Fig. 4C). This confirmed that TOCP treatment does result in a retraction/degeneration of established neurites in our model.

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