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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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Mmp1 processes the PDF neuropeptide in vitro.A–D. Reverse-phase HPLC profiles of Mmp1 alone (A), PDF alone (B), PDF+Mmp1 (C) or PDF+Mmp1+Batimastat (D) incubated for 1 h at 37°C. C. Peaks 1 through 4 contained PDF fragments and the peak 5 was full-length PDF as determined by MS/MS shown in Table 1. D. Note the absence of PDF degradation products when Mmp1 was preincubated with the MMP inhibitor Batimastat. Fractions 6 and 7 included PDF 1–19 as identified by MS/MS shown in Table 1. E. Schematic representation of Mmp1 preferred cleavage sites within PDF. Arrows indicate the peptide bonds hydrolyzed by Mmp1 as determined by MS/MS analysis. In bold and italics, P1' residues.
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pgen-1004700-g005: Mmp1 processes the PDF neuropeptide in vitro.A–D. Reverse-phase HPLC profiles of Mmp1 alone (A), PDF alone (B), PDF+Mmp1 (C) or PDF+Mmp1+Batimastat (D) incubated for 1 h at 37°C. C. Peaks 1 through 4 contained PDF fragments and the peak 5 was full-length PDF as determined by MS/MS shown in Table 1. D. Note the absence of PDF degradation products when Mmp1 was preincubated with the MMP inhibitor Batimastat. Fractions 6 and 7 included PDF 1–19 as identified by MS/MS shown in Table 1. E. Schematic representation of Mmp1 preferred cleavage sites within PDF. Arrows indicate the peptide bonds hydrolyzed by Mmp1 as determined by MS/MS analysis. In bold and italics, P1' residues.

Mentions: To further investigate the ability of Mmp1 to process or degrade PDF, the Mmp1 catalytic domain was expressed in E. coli as a His fusion protein. After fast protein liquid chromatography (FPLC) purification and refolding, Mmp1 activity on a previously characterized substrate was confirmed (Figure S5 A and [38]). Next, we incubated purified recombinant Mmp1 with PDF for 5 to 60 minutes at 37°C. The reaction products were purified by reverse-phase HPLC [39]. In contrast to recombinant Mmp1 (Figure 5A) and PDF alone (Figure 5B), co-incubation of PDF with Mmp1 gave rise to four novel peaks consistent with PDF fragments (Figure 5Cand Figures S6 A–B). Moreover, preincubation of Mmp1 with Batimastat, a well-characterized inhibitor of mammalian metalloproteinases [40], prevented PDF cleavage, underscoring that Mmp1 (as opposed to any contaminant potentially present in the original purified fraction) specifically hydrolyzes the neuropeptide (Figure 5D). To identify Mmp1 cleavage sites, the four degradation peaks were analyzed by MALDI-TOF-TOF. In the fast eluting fraction, peptides containing the C-terminal sequence of PDF (corresponding to the fragment LSLPKNMNDA of the reported sequence [41]) and to the fragment LLSLPKNMNDA were identified (Table 1). Additional fractions included peptides containing the N-terminal PDF sequence (corresponding to amino-acids YNSELINSL), thereby identifying the P1' L-L and P1' L-S as primary sites of Mmp1 cleavage (Figure 5E). We also tested whether Mmp2 could degrade PDF in vitro. Surprisingly, no novel peaks were detected upon incubation under the same conditions that resulted in Mmp1-directed degradation, even though Mmp2 was able to degrade a previously reported fluorogenic substrate for Mmp2 [42], thus confirming that recombinant Mmp2 displays proteolytic activity (Figure S5 B–C).


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)

Mmp1 processes the PDF neuropeptide in vitro.A–D. Reverse-phase HPLC profiles of Mmp1 alone (A), PDF alone (B), PDF+Mmp1 (C) or PDF+Mmp1+Batimastat (D) incubated for 1 h at 37°C. C. Peaks 1 through 4 contained PDF fragments and the peak 5 was full-length PDF as determined by MS/MS shown in Table 1. D. Note the absence of PDF degradation products when Mmp1 was preincubated with the MMP inhibitor Batimastat. Fractions 6 and 7 included PDF 1–19 as identified by MS/MS shown in Table 1. E. Schematic representation of Mmp1 preferred cleavage sites within PDF. Arrows indicate the peptide bonds hydrolyzed by Mmp1 as determined by MS/MS analysis. In bold and italics, P1' residues.
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Related In: Results  -  Collection

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pgen-1004700-g005: Mmp1 processes the PDF neuropeptide in vitro.A–D. Reverse-phase HPLC profiles of Mmp1 alone (A), PDF alone (B), PDF+Mmp1 (C) or PDF+Mmp1+Batimastat (D) incubated for 1 h at 37°C. C. Peaks 1 through 4 contained PDF fragments and the peak 5 was full-length PDF as determined by MS/MS shown in Table 1. D. Note the absence of PDF degradation products when Mmp1 was preincubated with the MMP inhibitor Batimastat. Fractions 6 and 7 included PDF 1–19 as identified by MS/MS shown in Table 1. E. Schematic representation of Mmp1 preferred cleavage sites within PDF. Arrows indicate the peptide bonds hydrolyzed by Mmp1 as determined by MS/MS analysis. In bold and italics, P1' residues.
Mentions: To further investigate the ability of Mmp1 to process or degrade PDF, the Mmp1 catalytic domain was expressed in E. coli as a His fusion protein. After fast protein liquid chromatography (FPLC) purification and refolding, Mmp1 activity on a previously characterized substrate was confirmed (Figure S5 A and [38]). Next, we incubated purified recombinant Mmp1 with PDF for 5 to 60 minutes at 37°C. The reaction products were purified by reverse-phase HPLC [39]. In contrast to recombinant Mmp1 (Figure 5A) and PDF alone (Figure 5B), co-incubation of PDF with Mmp1 gave rise to four novel peaks consistent with PDF fragments (Figure 5Cand Figures S6 A–B). Moreover, preincubation of Mmp1 with Batimastat, a well-characterized inhibitor of mammalian metalloproteinases [40], prevented PDF cleavage, underscoring that Mmp1 (as opposed to any contaminant potentially present in the original purified fraction) specifically hydrolyzes the neuropeptide (Figure 5D). To identify Mmp1 cleavage sites, the four degradation peaks were analyzed by MALDI-TOF-TOF. In the fast eluting fraction, peptides containing the C-terminal sequence of PDF (corresponding to the fragment LSLPKNMNDA of the reported sequence [41]) and to the fragment LLSLPKNMNDA were identified (Table 1). Additional fractions included peptides containing the N-terminal PDF sequence (corresponding to amino-acids YNSELINSL), thereby identifying the P1' L-L and P1' L-S as primary sites of Mmp1 cleavage (Figure 5E). We also tested whether Mmp2 could degrade PDF in vitro. Surprisingly, no novel peaks were detected upon incubation under the same conditions that resulted in Mmp1-directed degradation, even though Mmp2 was able to degrade a previously reported fluorogenic substrate for Mmp2 [42], thus confirming that recombinant Mmp2 displays proteolytic activity (Figure S5 B–C).

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