Organophosphate-Induced Changes in the PKA Regulatory Function of Swiss Cheese/NTE Lead to Behavioral Deficits and Neurodegeneration

<div><p>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 <i>Drosophila</i>, 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.</p></div

Effects of SWS levels on TOCP-induced behavioral deficits.

<p><b>A</b>. Wild type flies treated with 8 mg/ml or 16 mg/ml TOCP show a significantly reduced performance in the fast phototaxis assay. <b>B</b>. A similar deficit is detectable in 14 d old control flies (expressing lacZ pan-neuronally; <i>elav</i>>lacZ) treated with 8 mg/ml TOCP. Expressing additional SWS pan-neuronally (<i>elav</i>>SWS) does not protect 14 d old flies from behavioral deficits caused by TOCP however heterozygosity for <i>sws¹</i> (<i>sws</i>/WT) protects flies from the TOCP induced behavioral deficits (treated WT to <i>sws</i>/WT, *p<0.05). <b>C</b>. Comparing untreated flies in the phototaxis assay shows that overexpression of SWS alone results in less successful transitions already in 7 d old flies. This effect is even more severe when untreated 14 d old flies are tested. The analysis in <b>A</b> was done using one-way ANOVA with a Dunett's post test and the analyses in <b>B, C</b> with a Student's t-test, comparing treated and untreated flies of each genotype (in <b>B</b>) and control and SWS overexpressing flies of a given age (in C). n = is number of groups tested with 10–20 flies each. All flies were females. SEMs are indicated in all graphs. *p<0.05, ***p<0.001. (The was no significant difference in the variance in any of the comparisons).</p

TOCP induces neurite shortening in primary neuronal cultures.

<p><b>A</b>. 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>B</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. <b>C</b>. 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>B</b> = 2 µm. (The variances were significantly different between treated and untreated cells; p<0.001).</p

PKA-C3 and SWSR^133A expression prevent TOCP-induced reduction in PKA activity.

<p><b>A</b>. PKA-C3 overexpression increases PKA activity and this is not affected by TOCP treatment. <b>B</b>. Additional expression of SWS significantly reduces PKA activity whereas heterozygosity for <i>sws¹</i> does not. The values are shown in percent of the PKA activity in untreated wild type. n = number of independent measurement and the SEMs are indicated. Student's t-tests were used to compare activity in untreated and treated flies. One-way ANOVA with a Dunett's post test was used to compare the untreated flies in B. *p<0.05, ***p<0.001. (.The variances were not significantly different).</p

PKA-C3 overexpression protects against TOCP-induced degeneration and behavioral deficits.

<p><b>A</b>. Flies expressing additional PKA-C3 in neurons via <i>elav</i>-GAL4 do not show the TOCP-induced reduction in performance seen in <i>elav</i>>lacZ control flies. Also flies expressing the PKA-C3 binding deficient SWS^R133A 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 (<i>elav</i>>SWS). <b>B</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. <b>C</b>. 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 <b>A</b>, n = number of cells or head sections analyzed in <b>B</b> and <b>C</b>. Student's t-tests were used to compare treated and untreated flies and to compare untreated SWS and SWSR^133A overexpressing flies. A student's t-test was also used to compare vacuole size in untreated PKA-C3 overexpresing flies with controls in <b>B</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).</p

SWS levels do not affect axonal shortening.

<p><b>A</b>. Neurite length was significantly shorter in all TOCP treated (14 µg/ml) cells and SWS levels had no significant effect on this TOCP-induced phenotype. <b>B</b>. Paraoxon (PO, 4 µg/ml) treatment resultin similar neurite shortening as TOCP treatment. A Student's t-test was used to compare treated and untreated cells of each genotype in <b>A</b>. One-way ANOVA and Dunett's post test was used for <b>B</b>. n = number of cells analyzed and SEMs are indicated. ***p<0.001. (The variances were significantly different between treated and untreated cells; p<0.001).</p

TOCP treated flies show neuronal degeneration.

<p><b>A</b>. A paraffin head section of a wild type fly 14 d after vehicle treatment does not show overt degeneration. <b>B</b>. In contrast, a few vacuoles (arrowheads) have formed in an age-matched fly treated with 16 mg/ml TOCP. <b>C</b>. Measuring the area of vacuoles in the brain revealed a significant increase in 14 d old flies treated with 8 mg/ml or 16 mg/ml TOCP. Analysis was done using one-way ANOVA with a Dunett's post test to compare to vehicle treated flies. SEMs are indicated, n = number of flies analyzed; **p<0.01, ***p<0.001. Scale bar in <b>B</b> = 2 µm. re = retina, la = lamina, me = medulla, lo = lobila, lp = lobula plate. Scale bar in <b>A</b> = 40 µm. (The variance was not significantly different).</p

Loss of PKA-C3 causes behavioral and degenerative phenotypes.

<p><b>A</b>. Inducing a UAS-PKA-C3^RNAi construct pan-neuronally with <i>Appl</i>-GAL4 (<i>Appl</i>>PKA-C3^RNAi) did not result in a significant reduction in performance compared to controls (<i>Appl</i>>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>B</b>. Measuring the vacuole area in 14 d old <i>Appl</i>>dcr;PKA-C3^RNAi flies did not reveal a significant difference to age-matched <i>Appl</i>>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. <b>C</b>. 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 SWS^R133A. 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.</p

TOCP treatment inhibits SWS-esterase activity, but not AChE activity.

<p><b>A</b>. Esterase activity against phenyl valerate is significantly inhibited by TOCP treatment (16 mg/ml) in wild type whereas the residual activity in <i>sws¹</i> is not further reduced by TOCP. Flies overexpressing SWS using <i>elav</i>-GAL4 show approximately a 1.6 fold increase in activity in vehicle treated flies compared to wild type. Although this activity is significantly reduced by TOCP, the SWS overexpressing flies still show 50% of the activity of untreated wild type. Two independent measurements were done for treated flies and four for untreated flies. <b>B</b>. Neither TOCP treatment nor SWS levels have a significant effect on AChE activity. Two independent measurements were performed for each genotype and treatment. <b>A, B</b>. Flies were tested at the end of the 16 h treatment period. All values are shown relative to untreated wild type. A Student's t-test was used to compare each treated group to its corresponding untreated one. SEMs are indicated; **p<0.01. (The variances were not significantly different between treated and untreated flies for each genotype).</p

TOCP treatment induces lethality.

<p><b>A</b>. Wild type fly fed on glucose. <b>B</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). <b>C</b>. 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)</p