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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

Mmps are key players of the structural plasticity of PDF neurons.A. Schematic diagram illustrating the standard protocol and method for the analysis of the complexity of the PDF axonal arbor on confocal images. In all figures “VEH” and “RU” stand for “vehicle”- and “RU486”-containing fly food. B. Adult-specific Mmp overexpression triggers structural phenotypes. Left panel. Representative confocal images of GFP immunoreactivity at the dorsal protocerebrum at the early subjective day (CT2) and early subjective night (CT14) during the 4th day of constant darkness (DD4). Right panel. Quantitation of total axonal crosses. Wild type flies display circadian structural remodeling of axonal terminals while animals overexpressing Mmp1 or Mmp2 show reduced and constant complexity. Throughout the manuscript all experimental groups include CD8GFP, so the control group “+” refers to a single copy of CD8GFP;pdf-GS. Throughout the manuscript the average ± standard error of the mean is shown. C. Adult-specific Mmp downregulation also affects dorsal axonal branches. Silencing either Mmp1 or Mmp2 abolished circadian structural plasticity leading to a more complex structure clamped at the daytime configuration. Data represents the average of 4 to 5 experiments and a minimum of 27 brains were analyzed per CT/Genotype. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). For more details, see the Statistics section in Materials and Methods. “+” refers to a single copy of the pdf-GS/CD8GFP;Dcr2 transgenes. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.
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pgen-1004700-g001: Mmps are key players of the structural plasticity of PDF neurons.A. Schematic diagram illustrating the standard protocol and method for the analysis of the complexity of the PDF axonal arbor on confocal images. In all figures “VEH” and “RU” stand for “vehicle”- and “RU486”-containing fly food. B. Adult-specific Mmp overexpression triggers structural phenotypes. Left panel. Representative confocal images of GFP immunoreactivity at the dorsal protocerebrum at the early subjective day (CT2) and early subjective night (CT14) during the 4th day of constant darkness (DD4). Right panel. Quantitation of total axonal crosses. Wild type flies display circadian structural remodeling of axonal terminals while animals overexpressing Mmp1 or Mmp2 show reduced and constant complexity. Throughout the manuscript all experimental groups include CD8GFP, so the control group “+” refers to a single copy of CD8GFP;pdf-GS. Throughout the manuscript the average ± standard error of the mean is shown. C. Adult-specific Mmp downregulation also affects dorsal axonal branches. Silencing either Mmp1 or Mmp2 abolished circadian structural plasticity leading to a more complex structure clamped at the daytime configuration. Data represents the average of 4 to 5 experiments and a minimum of 27 brains were analyzed per CT/Genotype. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). For more details, see the Statistics section in Materials and Methods. “+” refers to a single copy of the pdf-GS/CD8GFP;Dcr2 transgenes. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.

Mentions: To examine a possible contribution of Mmps to the circadian structural plasticity of the sLNvs axonal terminals we altered Mmp1 or Mmp2 expression specifically in PDF neurons and analyzed the degree of arborization at the dorsal protocerebrum at two time points during the subjective day, at Circadian Time 2 (CT2, 2 hours after the lights should have been on) and CT14 (2 hours after lights should have been off) (Figure 1A). We restricted our treatment to the adult stage by using the pdf-GS RU486-inducible GeneSwitch strain recently described [30] to bypass any potential developmental effect. As previously described [14], control flies displayed a more complex arborization pattern during the early subjective day (CT2) and less arborized display during the early subjective night (CT14). On the contrary, adult-specific Mmp1 or Mmp2 overexpression in PDF neurons abolished any remodeling of dorsal projections, leading to a non-oscillating and less complex circuit that shows even fewer axonal crosses than the nighttime control neurons (Figure 1B). Overexpression with independent transgenic lines rendered similar results (Figure S1 A). A more detailed analysis of structural complexity indicated that Mmp1 does not affect its total axonal length while it does reduce the complexity of the arborizations all along the axonal projections. In contrast, Mmp2 has a significant effect on total axonal length indicating that the changes trigged by Mmp2 overexpression involve modification of the length of axonal terminals (Figure S1 B–D). Thus, although both Mmps impact the circadian remodeling of PDF neurons, the underlying mechanisms are not necessarily the same.


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)

Mmps are key players of the structural plasticity of PDF neurons.A. Schematic diagram illustrating the standard protocol and method for the analysis of the complexity of the PDF axonal arbor on confocal images. In all figures “VEH” and “RU” stand for “vehicle”- and “RU486”-containing fly food. B. Adult-specific Mmp overexpression triggers structural phenotypes. Left panel. Representative confocal images of GFP immunoreactivity at the dorsal protocerebrum at the early subjective day (CT2) and early subjective night (CT14) during the 4th day of constant darkness (DD4). Right panel. Quantitation of total axonal crosses. Wild type flies display circadian structural remodeling of axonal terminals while animals overexpressing Mmp1 or Mmp2 show reduced and constant complexity. Throughout the manuscript all experimental groups include CD8GFP, so the control group “+” refers to a single copy of CD8GFP;pdf-GS. Throughout the manuscript the average ± standard error of the mean is shown. C. Adult-specific Mmp downregulation also affects dorsal axonal branches. Silencing either Mmp1 or Mmp2 abolished circadian structural plasticity leading to a more complex structure clamped at the daytime configuration. Data represents the average of 4 to 5 experiments and a minimum of 27 brains were analyzed per CT/Genotype. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). For more details, see the Statistics section in Materials and Methods. “+” refers to a single copy of the pdf-GS/CD8GFP;Dcr2 transgenes. 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-g001: Mmps are key players of the structural plasticity of PDF neurons.A. Schematic diagram illustrating the standard protocol and method for the analysis of the complexity of the PDF axonal arbor on confocal images. In all figures “VEH” and “RU” stand for “vehicle”- and “RU486”-containing fly food. B. Adult-specific Mmp overexpression triggers structural phenotypes. Left panel. Representative confocal images of GFP immunoreactivity at the dorsal protocerebrum at the early subjective day (CT2) and early subjective night (CT14) during the 4th day of constant darkness (DD4). Right panel. Quantitation of total axonal crosses. Wild type flies display circadian structural remodeling of axonal terminals while animals overexpressing Mmp1 or Mmp2 show reduced and constant complexity. Throughout the manuscript all experimental groups include CD8GFP, so the control group “+” refers to a single copy of CD8GFP;pdf-GS. Throughout the manuscript the average ± standard error of the mean is shown. C. Adult-specific Mmp downregulation also affects dorsal axonal branches. Silencing either Mmp1 or Mmp2 abolished circadian structural plasticity leading to a more complex structure clamped at the daytime configuration. Data represents the average of 4 to 5 experiments and a minimum of 27 brains were analyzed per CT/Genotype. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan post-hoc test). For more details, see the Statistics section in Materials and Methods. “+” refers to a single copy of the pdf-GS/CD8GFP;Dcr2 transgenes. In both experiments all the experimental groups include RU to induce expression. Scale: 10 µm.
Mentions: To examine a possible contribution of Mmps to the circadian structural plasticity of the sLNvs axonal terminals we altered Mmp1 or Mmp2 expression specifically in PDF neurons and analyzed the degree of arborization at the dorsal protocerebrum at two time points during the subjective day, at Circadian Time 2 (CT2, 2 hours after the lights should have been on) and CT14 (2 hours after lights should have been off) (Figure 1A). We restricted our treatment to the adult stage by using the pdf-GS RU486-inducible GeneSwitch strain recently described [30] to bypass any potential developmental effect. As previously described [14], control flies displayed a more complex arborization pattern during the early subjective day (CT2) and less arborized display during the early subjective night (CT14). On the contrary, adult-specific Mmp1 or Mmp2 overexpression in PDF neurons abolished any remodeling of dorsal projections, leading to a non-oscillating and less complex circuit that shows even fewer axonal crosses than the nighttime control neurons (Figure 1B). Overexpression with independent transgenic lines rendered similar results (Figure S1 A). A more detailed analysis of structural complexity indicated that Mmp1 does not affect its total axonal length while it does reduce the complexity of the arborizations all along the axonal projections. In contrast, Mmp2 has a significant effect on total axonal length indicating that the changes trigged by Mmp2 overexpression involve modification of the length of axonal terminals (Figure S1 B–D). Thus, although both Mmps impact the circadian remodeling of PDF neurons, the underlying mechanisms are not necessarily the same.

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