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Mmp1 processing of the PDF neuropeptide regulates circadian structural plasticity of pacemaker neurons.

Depetris-Chauvin A, Fernández-Gamba A, Gorostiza EA, Herrero A, Castaño EM, Ceriani MF - PLoS Genet. (2014)

Bottom Line: Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections.However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior.These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas-Buenos Aires (IIB-BA, CONICET), Buenos Aires, Argentina.

ABSTRACT
In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

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Related in: MedlinePlus

Cell autonomous Mmp1 expression regulates PDF levels.A. Overexpression experiments Left panel. Representative confocal images of PDF immunoreactivity at the dorsal protocerebrum taken during CT2 and CT14 on DD4. Right panel. PDF levels at the dorsal protocerebrum. Control flies exhibit circadian oscillation of PDF levels, while Mmp1 overexpression reduces PDF to levels lower than those observed at nighttime in controls. In contrast, Mmp2 overexpression leads to intermediate levels. “+” in the x axis refers to a single copy of CD8GFP; pdf-GS. B. Downregulation experiments. Reducing Mmp1 but not Mmp2 levels abolishes circadian oscillations in PDF immunoreactivity to constant daytime levels. “+” in the x axis refers to a single copy of CD8GFP, Dcr2; pdf-GS. Data represents the average (± standard error of the mean) of at least 3 independent experiments and a minimum of 23 flies per Genotype/CT were analyzed. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). In overexpression experiments logarithmic transformation was applied to fulfill ANOVA requirements. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.
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pgen-1004700-g003: Cell autonomous Mmp1 expression regulates PDF levels.A. Overexpression experiments Left panel. Representative confocal images of PDF immunoreactivity at the dorsal protocerebrum taken during CT2 and CT14 on DD4. Right panel. PDF levels at the dorsal protocerebrum. Control flies exhibit circadian oscillation of PDF levels, while Mmp1 overexpression reduces PDF to levels lower than those observed at nighttime in controls. In contrast, Mmp2 overexpression leads to intermediate levels. “+” in the x axis refers to a single copy of CD8GFP; pdf-GS. B. Downregulation experiments. Reducing Mmp1 but not Mmp2 levels abolishes circadian oscillations in PDF immunoreactivity to constant daytime levels. “+” in the x axis refers to a single copy of CD8GFP, Dcr2; pdf-GS. Data represents the average (± standard error of the mean) of at least 3 independent experiments and a minimum of 23 flies per Genotype/CT were analyzed. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). In overexpression experiments logarithmic transformation was applied to fulfill ANOVA requirements. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.

Mentions: PDF is crucial for the proper control of circadian locomotor activity since pdfo1 and pdf Receptor (pdfR/han) mutants largely become arrhythmic under DD conditions [5], [34],[35]. Therefore, we wondered if the behavioral phenotypes described for flies with Mmp1 missexpression were reflecting an alteration of PDF signaling. To address this possibility we measured the levels of the neuropeptide at the dorsal protocerebrum by immunohistochemistry during the early subjective day (CT2) and night (CT14). In control animals, PDF immunoreactivity changes at the dorsal terminals, with high levels at CT2 and low levels at CT14 (Figure 3 A–B, upper panels). Overexpression of Mmp1 or Mmp2 affected PDF immunoreactivity and disrupted its circadian oscillation. Mmp1 effect was far more severe, resulting in reduced PDF levels at both timepoints to an extent that reached statistical significance; on the contrary, Mmp2 affected PDF levels rather subtly and led to intermediate levels that did not significantly differ from any timepoint in control flies (Figure 3A). Overexpression with independent transgenic lines retrieved similar results (Figure S4 A). RNAi analysis showed that reduced Mmp1 but not Mmp2 levels abolished the circadian oscillation in PDF immunoreactivity, resulting in levels reminiscent of the daytime configuration (Figure 3B). Interestingly, the fact that downregulation of Mmp1 but not Mmp2 affects PDF immunoreactivity correlates with the specific effect of silencing Mmp1 on locomotor activity, suggesting that clamping PDF at high levels might be the cause of the behavioral phenotypes observed.


Mmp1 processing of the PDF neuropeptide regulates circadian structural plasticity of pacemaker neurons.

Depetris-Chauvin A, Fernández-Gamba A, Gorostiza EA, Herrero A, Castaño EM, Ceriani MF - PLoS Genet. (2014)

Cell autonomous Mmp1 expression regulates PDF levels.A. Overexpression experiments Left panel. Representative confocal images of PDF immunoreactivity at the dorsal protocerebrum taken during CT2 and CT14 on DD4. Right panel. PDF levels at the dorsal protocerebrum. Control flies exhibit circadian oscillation of PDF levels, while Mmp1 overexpression reduces PDF to levels lower than those observed at nighttime in controls. In contrast, Mmp2 overexpression leads to intermediate levels. “+” in the x axis refers to a single copy of CD8GFP; pdf-GS. B. Downregulation experiments. Reducing Mmp1 but not Mmp2 levels abolishes circadian oscillations in PDF immunoreactivity to constant daytime levels. “+” in the x axis refers to a single copy of CD8GFP, Dcr2; pdf-GS. Data represents the average (± standard error of the mean) of at least 3 independent experiments and a minimum of 23 flies per Genotype/CT were analyzed. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). In overexpression experiments logarithmic transformation was applied to fulfill ANOVA requirements. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214601&req=5

pgen-1004700-g003: Cell autonomous Mmp1 expression regulates PDF levels.A. Overexpression experiments Left panel. Representative confocal images of PDF immunoreactivity at the dorsal protocerebrum taken during CT2 and CT14 on DD4. Right panel. PDF levels at the dorsal protocerebrum. Control flies exhibit circadian oscillation of PDF levels, while Mmp1 overexpression reduces PDF to levels lower than those observed at nighttime in controls. In contrast, Mmp2 overexpression leads to intermediate levels. “+” in the x axis refers to a single copy of CD8GFP; pdf-GS. B. Downregulation experiments. Reducing Mmp1 but not Mmp2 levels abolishes circadian oscillations in PDF immunoreactivity to constant daytime levels. “+” in the x axis refers to a single copy of CD8GFP, Dcr2; pdf-GS. Data represents the average (± standard error of the mean) of at least 3 independent experiments and a minimum of 23 flies per Genotype/CT were analyzed. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). In overexpression experiments logarithmic transformation was applied to fulfill ANOVA requirements. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.
Mentions: PDF is crucial for the proper control of circadian locomotor activity since pdfo1 and pdf Receptor (pdfR/han) mutants largely become arrhythmic under DD conditions [5], [34],[35]. Therefore, we wondered if the behavioral phenotypes described for flies with Mmp1 missexpression were reflecting an alteration of PDF signaling. To address this possibility we measured the levels of the neuropeptide at the dorsal protocerebrum by immunohistochemistry during the early subjective day (CT2) and night (CT14). In control animals, PDF immunoreactivity changes at the dorsal terminals, with high levels at CT2 and low levels at CT14 (Figure 3 A–B, upper panels). Overexpression of Mmp1 or Mmp2 affected PDF immunoreactivity and disrupted its circadian oscillation. Mmp1 effect was far more severe, resulting in reduced PDF levels at both timepoints to an extent that reached statistical significance; on the contrary, Mmp2 affected PDF levels rather subtly and led to intermediate levels that did not significantly differ from any timepoint in control flies (Figure 3A). Overexpression with independent transgenic lines retrieved similar results (Figure S4 A). RNAi analysis showed that reduced Mmp1 but not Mmp2 levels abolished the circadian oscillation in PDF immunoreactivity, resulting in levels reminiscent of the daytime configuration (Figure 3B). Interestingly, the fact that downregulation of Mmp1 but not Mmp2 affects PDF immunoreactivity correlates with the specific effect of silencing Mmp1 on locomotor activity, suggesting that clamping PDF at high levels might be the cause of the behavioral phenotypes observed.

Bottom Line: Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections.However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior.These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética del Comportamiento, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas-Buenos Aires (IIB-BA, CONICET), Buenos Aires, Argentina.

ABSTRACT
In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

Show MeSH
Related in: MedlinePlus