Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

<div><p>Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human <i>CPA6</i> gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in <i>CPA6</i> identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of <i>cpa6</i> mRNA in zebrafish (<i>Danio rerio</i>) larvae. Knockdown of <i>cpa6</i> mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of <i>cpa6</i> mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (<i>bdnf</i>, <i>npy</i>, <i>chga</i>, <i>pcsk1nl</i>, <i>tac1</i>, <i>nts</i>, <i>edn1</i>), a neuropeptide processing enzyme (<i>cpe</i>), transcription factor (<i>c-fos</i>), and molecules implicated in glutamatergic signaling (<i>grin1a</i> and <i>slc1a2b</i>). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for <i>c-fos</i> which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like <i>cpa6</i> knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first <i>in vivo</i> demonstration of the molecular and behavioral consequences of <i>cpa6</i> insufficiency.</p></div

Effects of pilocarpine on mRNA levels of signaling molecules in 3 dpf zebrafish embryos.

<p>Wild-type zebrafish embryos were exposed to 60 mM pilocarpine for 1 h, the fish were transferred to drug-free embryo medium, and mRNA was extracted after 1, 5, or 23 h washout without drug. Quantitative PCR was used to measure mRNA levels of targets. Error bars show SEM (n = 4 for each group). Statistical analysis was performed by Student’s <i>t</i> test: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 compared with respective Ctrl-MO group.</p

Knockdown of <i>cpa6</i> reduces PTZ-evoked convulsive swimming behavior in 3 dpf zebrafish embryos.

<p>(A) Zebrafish were tested for convulsive swimming behavior in the presence of 2.5 or 15 mM PTZ. After a 10 minute baseline period without drug, 2.5 or 15 mM PTZ were added to the bath and convulsive swimming behavior was recorded by an observer blinded to the treatment groups, using previously established criteria [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152905#pone.0152905.ref016" target="_blank">16</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152905#pone.0152905.ref017" target="_blank">17</a>]. Statistical analysis was performed by repeated measures ANOVA, followed by Tukey HSD: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 compared with No drug group in respective time points; #, <i>p</i> < 0.05 compared with 2.5 mM PTZ group in respective time points. (B) Quantitative PCR revealed near total knockdown in the targeted exon for CPA6-MO2, while co-injection of CPA6-MO2 and 500 pg mRNA increased transcript levels of <i>cpa6</i> by ~25 fold (n = 4). One-way ANOVA, followed by Tukey HSD: ***, <i>p</i> < 0.001 compared with Ctrl-MO group; #, <i>p</i> < 0.001 compared with CPA6-MO2 group. (C) Quantitative PCR revealed near-total knockdown of the targeted exon for CPA6-MO4 (n = 4). Student’s <i>t</i> test: ***, <i>p</i> < 0.001 compared with Ctrl-MO group. (D) CPA6-MO2 injected animals exposed to 2.5 mM PTZ showed fewer bouts of convulsive swimming behavior relative to Ctrl-MO and this behavior was rescued by co-injection of CPA6-MO2 and <i>cpa6</i> mRNA. Repeated measures ANOVA, followed by Tukey HSD: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 compared with Ctrl-MO group in respective time points. (E) CPA6-MO4 injected animals showed fewer bouts of convulsive swimming behavior relative to Ctrl-MO. Repeated measures ANOVA, followed by Tukey HSD: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 compared with Ctrl-MO group in respective time points. (F) The “no drug” control group of zebrafish embryos displayed no bouts of convulsive swimming behavior, resulting in a latency greater than the testing period (1080 seconds). Latency to the first bout was decreased in zebrafish embryos exposed to 2.5 and 15 mM PTZ. One-way ANOVA, followed by Tukey HSD: ***, <i>p</i> < 0.001 compared with No drug group; #, <i>p</i> < 0.05 compared with 2.5 mM PTZ group. (G, H) CPA6-MO2 and -MO4 injected embryos exposed to 2.5 mM PTZ had a higher latency to the first bout of convulsive swimming behavior when compared to fish injected with control morpholino. In panels G and H, the y-axis is similar to that of panel F. Co-injection of <i>cpa6</i> mRNA along with CPA6-MO2 blocked the effect of the morpholino oligonucleotide. One-way ANOVA, followed by Tukey HSD used in panel G: *, <i>p</i> < 0.05 compared with Ctrl-MO and CPA6-MO2 + <i>cpa6</i> mRNA groups. Student’s t test used in panel H: **, <i>p</i> < 0.01 compared with Ctrl-MO group. Error bars show SEM.</p

Zebrafish embryos injected with CPA6-MO2 and -MO4 are responsive to stimuli.

<p>Zebrafish embryos 3 dpf were stimulated by pushing 200 μl of the embryo media in which they were suspended towards them using a P-200 pipette at moderate pressure (A) or by touching with a hair (B). Water stimuli were calibrated to evoke a response only ~40% of the time under basal conditions while touch stimuli evoked a response 80% of the time. Statistical analysis was performed by one-way ANOVA. (C,D) PTZ (2.5 mM) was added to the embryo media and fish were tested at 5 minute intervals for responsiveness to the water stimulus. There were no statistically significant differences between embryos injected with Ctrl-MO, CPA6-MO2 or CPA6-MO4 either in basal conditions or in the presence of PTZ during the time periods examined for either stimulation paradigm. Statistical analysis was performed by repeated measures ANOVA. Error bars show SEM.</p

Knockdown of <i>cpa6</i> reduces pilocarpine-induced increase in locomotion in 3 dpf zebrafish embryos.

<p>(A) Animals were placed in a 12-well cluster plate and tested for number of crossings between quadrants (locomotor activity) in the presence of 15, 30 or 60 mM pilocarpine. After a 10 minute baseline period, pilocarpine was added to the bath to produce a final concentration of 15, 30 or 60 mM. The number of crossings was recorded over a 1.5 minute period by an observer blinded to the treatment group; for baseline the period from 8.5–10.0 min was analyzed, and for pilocarpine the period from 5.0–6.5 min after drug treatment was analyzed. The number of crossings was increased by pilocarpine, with a greater increase in zebrafish embryos injected with Ctrl-MO compared to CPA6-MO2. Statistical analysis was performed by two-way ANOVA, followed by Tukey HSD: **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 compared with respective Ctrl-MO group. (B) Quantitative PCR revealed near total knockdown in the targeted exon for CPA6-MO2 (n = 4). Student’s <i>t</i> test: ***, <i>p</i> < 0.001 compared with Ctrl-MO group. (C) CPA6-MO2 injected animals exposed to 15 mM pilocarpine showed decreased number of crossings relative to Ctrl-MO and this effect was partially rescued by co-injection of 500 pg <i>cpa6</i> mRNA along with the CPA6-MO2. One-way ANOVA, followed by Tukey HSD: *, <i>p</i> < 0.05; ***, <i>p</i> < 0.001 compared with Ctrl-MO group; #, <i>p</i> < 0.05 compared with CPA6-MO2 group. (D) Quantitative PCR revealed near total knockdown of the targeted exon for CPA6-MO2, while co-injection of <i>cpa6</i> mRNA along with CPA6-MO2 increased the transcript levels of <i>cpa6</i> (n = 4). One-way ANOVA followed by Tukey HSD: ***, <i>p</i> < 0.001 compared with Ctrl-MO group; #, <i>p</i> < 0.001 compared with CPA6-MO2 group. (E) CPA6-MO4 injected animals exposed to 15 mM pilocarpine showed decreased number of crossings relative to Ctrl-MO (n = 4). Student’s <i>t</i> test: ***, <i>p</i> < 0.001 compared with Ctrl-MO group. (F) Quantitative PCR revealed near total knockdown in the targeted exon for CPA6-MO4. Student’s <i>t</i> test: ***, <i>p</i> < 0.001 compared with Ctrl-MO group. Error bars show SEM.</p

PTZ treatment affects mRNA levels in zebrafish embryos.

<p>Zebrafish embryos (wild-type 3 dpf) were exposed to 15 mM PTZ for 1 h and then transferred to drug-free embryo medium for 1, 5, or 23 h after which mRNA was extracted. Quantitative PCR was used to examine levels of various mRNAs. Error bars show SEM (n = 4 for each group). Statistical analysis was performed by Student’s <i>t</i> test: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001 compared with Ctrl-MO group.</p

The response of 4 dpf zebrafish embryos to PTZ and pilocarpine is affected by a 1 h exposure to 60 mM pilocarpine at 3 dpf.

<p>Wild-type zebrafish embryos were divided into two groups. After 3 dpf, one group was exposed to 60 mM pilocarpine for 1 h while the other group was not treated with drug. Following an additional 23 h period, the 4 dpf zebrafish were examined for convulsive swimming behavior by an investigator blinded to the treatment groups. (A) In the absence of additional drug, no bouts of convulsive swimming behavior were observed over a 10 minute period for either unexposed or pilocarpine-exposed animals. When treated with 2.5 mM PTZ for 17 minutes, convulsive swimming behavior was observed. The group previously exposed to pilocarpine showed fewer bouts than the control group. Statistical analysis was performed by repeated measures ANOVA, followed by Tukey HSD: *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 compared with No pretreatment group in respective time points. (B) Latency to the first bout was measured after 2.5 mM PTZ exposure. Pre-treatment with 60 mM pilocarpine increased the latency to first bout: Student’s <i>t</i> test. **, <i>p</i> < 0.01 compared with no pretreatment group. (C) Animals were tested for number of crossings (locomotor activity) under baseline conditions and in response to 15 mM pilocarpine. No significant differences in number of crossings were observed in the group previously exposed to 60 mM pilocarpine, relative to control group; both groups showed a comparable increase in locomotion in response to 15 mM pilocarpine. Statistical analysis was performed by Student’s <i>t</i> test. Error bars show SEM.</p

Mn induces oxidative stress in the striatum.

<p>Oxidative stress was analyzed by DCF fluorescence in the striatum of young rats treated with Mn. The graphic shows the DCF fluorescence from rats treated for five days (PN8-12) with saline (control; NaCl 0.9%) or MnCl₂ at doses of 5, 10 or 20 mg/kg/day. The structures were analyzed on PN14. The data are expressed as percentage of the control and the values are mean ± S.E.M derived from eight independent experiments. Statistical analysis was performed by ANOVA followed by Duncan's test. ** p<0.01, *** p<0.001 compared to control.</p

Immature rats' weight gain exposed to Mn <i>in vivo</i>.

<p>Immature rats were treated with saline (control; NaCl 0.9%) or MnCl₂ at doses of 5, 10 or 20 mg/kg for five days (PN8-12). Body weights were measured throughout the treatment, and the weight gain recorded on PN14. Results represent mean ± S.E.M derived from eighteen independent experiments and are expressed in grams (g). Statistical analysis was performed by ANOVA followed by Duncan's test.</p>*<p>p<0.05 compared to control.</p

Protective effect of antioxidant Trolox™ on body weight gain in immature rats exposed to Mn <i>in vivo</i>.

<p>Immature rats were treated with saline (control; NaCl 0.9%), MnCl₂ 20 mg/kg (Mn), Trolox™ 1 mg/kg (Tr) or MnCl₂ 20 mg/kg plus Trolox™ 1 mg/kg (Mn + Tr) for five days (PN8-12). Body weights were measured throughout the treatment, and the weight gain recorded on PN14. Results represent mean ± S.E.M derived from nine independent experiments and are expressed in grams (g). Statistical analysis was performed by ANOVA followed by Duncan's test.</p>***<p>p<0.001 compared to control;</p>##<p>p<0.01 compared to Mn 20 mg/kg group.</p