Cell autonomous Mmp1 expression regulates PDF levels.

<p><b>A.</b> Overexpression experiments <b>Left panel.</b> Representative confocal images of PDF immunoreactivity at the dorsal protocerebrum taken during CT2 and CT14 on DD4. <b>Right panel.</b> 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 <i>CD8GFP</i>; <i>pdf</i>-GS. <b>B.</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 <i>CD8GFP</i>, <i>Dcr2</i>; <i>pdf</i>-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 <i>post-hoc</i> 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.</p

A model for the regulation of circadian axonal remodeling of sLNv neurons.

<p>The bidirectional arrow between electrical activity and Mmp1 suggests a possible coordination of both processes. Mmp1 effects on structural plasticity are dependent on the modulation of PDF levels at the sLNv terminals, via direct proteolysis, while Mmp2 appears to act downstream of the neuropeptide. Electrical activity regulates the overall level of complexity but it is not required to determine the circadian aspect of this remodeling. Given our current understanding Fas2 and EcR could act either upstream or downstream of PDF; however, the well-known Fas2 function points to a more direct modulation of circuit structure. Changes in the size of “PDF” and “Mmp1” molecules illustrate oscillations in abundance along the day.</p

Mmp1 modulates behavioral rhythmicity.

<p><b>A.</b> Representative actograms (<b>left panel</b>) and quantitation of percentage of rhythmicity (<b>right panel</b>) from overexpression experiments. Locomotor activity of individual flies was recorded for 4 days under light-dark cycles and then transferred to constant darkness (gray area) for 9 additional days. Overexpression of Mmp1 or Mmp2 with one UAS copy does not affect circadian locomotor activity. “+” in the x axis refers to a single copy of <i>CD8GFP</i>; <i>pdf</i>-GS. NS, non significant. <b>B.</b> Adult-specific Mmp downregulation trigger opposite effects on locomotor rhythmicity. Silencing Mmp1 but not Mmp2 alters normal circadian locomotor activity. “+” in the x axis refers to a single copy of <i>CD8GFP</i>; <i>pdf</i>-GS. Data represents at least 3 independent experiments and a minimum of 32 flies per Genotype/Condition were analyzed. Different letters indicate statistically significant differences with a p<0.05 (Two-way ANOVA with a Duncan <i>post-hoc</i> test). For other controls and measurements of endogenous period see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004700#pgen.1004700.s007" target="_blank">Table S1</a>.</p

PDF defines the axonal remodeling of its own neurons.

<p><b>A.</b> Quantitation of total axonal crosses from UAS-PDF rescue experiments. Overexpression of PDF rescues the structural plasticity defects caused by Mmp1 overexpression. “+” in the x axis refers to a single copy of <i>CD8GFP</i>; <i>pdf</i>-GS. Data represents the average (± standard error of the mean) between 3–5 independent experiments and a minimum of 21 flies were analyzed per Genotype/CT. <b>B.</b> PDF downregulation prevents circadian axonal remodeling of sLNv terminals and reduces daytime complexity to nighttime levels. “+” in the x axis refers to a single copy of <i>CD8GFP</i>, <i>Dcr2</i>; <i>pdf</i>-GS. Data represents the average (± standard error of the mean) between 3 independent experiments and a minimum of 25 flies were analyzed per Genotype/CT. In both experiments different letters indicate statistical differences with a p<0.05 (Two-way ANOVA with a Duncan <i>post-hoc</i> test) and all the experimental groups include RU to induce expression.</p

Mmp1 processes the PDF neuropeptide <i>in vitro</i>.

<p><b>A–D.</b> Reverse-phase HPLC profiles of Mmp1 alone (<b>A</b>), PDF alone (<b>B</b>), PDF+Mmp1 (<b>C</b>) or PDF+Mmp1+Batimastat (<b>D</b>) incubated for 1 h at 37°C. <b>C.</b> Peaks 1 through 4 contained PDF fragments and the peak 5 was full-length PDF as determined by MS/MS shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004700#pgen-1004700-t001" target="_blank">Table 1</a>. <b>D.</b> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004700#pgen-1004700-t001" target="_blank">Table 1</a>. <b>E.</b> 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.</p

Time-course, amount and pH-dependence of the formation of complexes between Aβ1-42 and IDEwt or IDEQ.

<p>(<b>A</b>) Western blots with anti-Aβ 6E10 showing the ∼120 kDa band corresponding to IDE-AβSCx (IDE-Aβ stable complex) as a function of the incubation time. Top panel, IDEQ; lower panel, IDEwt. Both PVDF membranes were developed simultaneously with a STORM 860 scanner. Below each Western blot, the same membranes stained with Coomassie blue, show IDEwt or IDEQ loading. (<b>B</b>) Densitometric data from Western blots for IDEQ (◯) and IDEwt (▴) were fitted to a single exponential equation using Graph Pad Prism v.4 software. Points represent the mean ± SEM from two independent experiments in duplicate. (<b>C</b>) IDEQ-AβSCx formation is partially competed by pre-incubation for 1 h with insulin at the indicated molar excess before the addition of Aβ1-42. Data are expressed as the percentage of the remaining Aβ-positive band at ∼120 kDa, in arbitrary units, as a function of insulin concentration. Each point represents the mean ± SEM of two independent experiments in duplicate. Inset: a representative Western blot of IDEQ-AβSCx developed with 6E10. (<b>D</b>) Densitometry of IDEQ-AβSCx at the indicated range of pH as determined by Western blot with anti-Aβ. Bars represent the mean ± SEM of three separate experiments. Inset: top, representative Western blot with anti-Aβ of IDEQ-AβSCx; bottom, Coomassie blue of IDEQ loaded in each lane. In panels (<b>A</b>), (<b>C</b>) and (<b>D</b>), IDEwt or IDEQ-AβSCxs are indicated by arrowheads.</p

Effect of IDEQ upon solubility and secondary structure of Aβ aggregates.

<p>(<b>A</b>) samples containing Aβ1-42 before incubation (“dead time”) and after incubation for 5 days with or without IDEQ were centrifuged at 3,000× g for 5 min and supernatants and pellets analyzed by Western blots with anti-Aβ 6E10 and 4G8. Arrowheads indicate: H, high molecular mass oligomers, T, Aβ tetramers and <i>t</i>, Aβ trimers. (<b>B</b>) Densitometric quantification of Aβ1-42 obtained from Western blots shown in panel (<b>A</b>). Bars represent the mean ± SEM of total Aβ immunoreactivity in arbitrary units (AU). * p<0.05, Student's <i>t</i> test. (<b>C</b>) Far UV-CD spectra recorded at “dead time” of Aβ1-42 (solid black line), IDEQ alone (dotted line) and Aβ1-42 co-incubated with IDEQ (solid gray line) at a 1∶300 molar ratio (IDEQ:Aβ). (<b>D</b>) Far UV-spectra recorded after 6 days of incubation of Aβ1-42 alone or Aβ1-42 with IDEQ at 1∶300 molar ratio. Samples were centrifuged as described above and supernatants analyzed. IDEQ alone, dotted line; Aβ1-42 alone, solid black line; Aβ1-42 co-incubated with IDEQ, solid gray line.</p

Aβ1-42 oligomers formed in the presence of IDEQ are not neurotoxic.

<p>(<b>A</b>) Representative AFM images showing the size and morphology of Aβ1-42 neurotoxic species. Left, Aβ1-42 incubated alone for 4 days. Approximately spherical species of ∼20–30 nm are indicated by arrowheads. Rods and short protofibrils are depicted by arrows. Inset: a larger Aβ1-42 protofibril is shown. Right; Aβ1-42 incubated in the presence of IDEQ at a 1∶10 molar ratio (IDEQ: Aβ) showing larger aggregates of 50–60 nm (arrowheads) and rods with lengths of ∼100–120 nm (arrows). (<b>B</b>) Representative immunofluorescence of primary differentiated neurons exposed to vehicle, Aβ1-42 alone or Aβ1-42 pre-incubated with IDEQ from top to bottom, as indicated. White arrows point at neuronal processes. Bars  = 30 μM. (<b>C</b>) Analysis of neuronal processes under the conditions as shown in panel (<b>A</b>). Bars represent the mean ± SEM of processes' lengths as measured from the centre of the neuronal body * p<0.01, one-way ANOVA, Tukey post-hoc test. (<b>D</b>) Viability of mature primary neurons after the indicated treatments as assessed by MTT reduction. * p<0.05, one-way ANOVA, Tukey post-hoc test. Results are shown for three independent experiments.</p

Effect of IDEQ and ATP upon the kinetic of Aβ aggregation and seeding.

<p>(<b>A</b>) Turbidity profiles of Aβ1-42 at 15 μM alone in working buffer (▴) or in the presence of IDEQ at the indicated molar ratios (IDEQ:Aβ), from top to bottom: 1∶200 (□), 1∶100 (Δ), 1∶10 (◯) and 1∶10 containing 0.5 mM ATP (<b>•</b>). Light scattering at 340 nm was measured every 30 min using a TECAN GENios multi-well reader (for clarity, only the points every other 90 min are shown). The bracket encloses the curves obtained after co-incubation of Aβ1-42 with IDEQ at the indicated conditions. Results are expressed as mean ± S.E.M. of at least two independent experiments in duplicate. (<b>B</b>) Representative TEM images of samples at steady state of Aβ1-42 alone (top) or with IDEQ at 1∶10 molar ratio in the presence of ATP (bottom). Bars = 100 nm. (<b>C</b>) Time course of Aβ1-42 aggregation alone (▴), in the presence of seeds previously formed with IDEQ (◯), or after the addition of pure Aβ1-42 seeds (□). (<b>D</b>) Kinetics of aggregation of Aβ1-42 after the addition of IDEQ (◯) or the same volume of working buffer (▴) to Aβ1-42 after 48 h of self-assembly, as indicated by the arrow.</p

IDEQ does not modify insulin conformation.

<p>(<b>A</b>) <i>Circular dichroism (CD) spectra</i> of 10 μM insulin (ins.) in working buffer (solid black line), insulin with IDEQ at 1∶100 molar ratio, enzyme:insulin (solid gray line) and IDEQ alone (dotted line) with no prior incubation. (<b>B</b>) Same samples as in panel (<b>A</b>) after incubation for 24 h at 25°C. Insulin alone (solid black line), insulin with IDEQ (solid gray line) and IDEQ alone (dotted line). (<b>C</b>) Western blot with anti-phospho-Akt and anti-total Akt of U-87 cell lysates. Cells were exposed for 30 min with insulin alone, insulin previously co-incubated with IDEwt or IDEQ, as indicated. Wortmannin (wort) was incubated at 10 nM for 30 min before treatments.</p