Role of White protein in the biosynthesis of <i>Drosophila</i> eye pigments and the neurotransmitters serotonin and dopamine.

<p>The White protein is an ABC transporter that, in combination with the Scarlet protein, transports tryptophan and, in combination with the Brown protein, guanine across the cell membrane into the cytoplasm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Ewart1" target="_blank">[7]</a>. Tryptophan is a precursor of the <i>Drosophila</i> Ommochrome pigment xanthommatin (brown) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Summers1" target="_blank">[6]</a>, but is also a precursor of the neurotransmitter serotonin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Murch1" target="_blank">[13]</a>, as illustrated on the left. Guanine is a precursor of tetrahydrobiopterin (BH₄), which in turn is a precursor of most Drosopterins (red eye pigments of <i>Drosophila</i>) but also an essential cofactor in the conversion of tyrosine to dopamine, as indicated on the right, and of tryptophan to serotonin, as depicted on the left <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Visser1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Thny1" target="_blank">[12]</a>.</p

Immunohistochemical analysis of the localization of the Reelin proteases in the hippocampus of 3xTg-AD mice and their <i>non-transgenic</i> controls.

<p>(<b>A–C</b>) Immunoperoxidase labeling using anti-tPA (<b>A</b>), anti-ADAMTS-4 (<b>B</b>), and anti-ADAMTS-5 antibodies (<b>C</b>). (<b>D</b>) Semi-quantitative analysis of the tPA, ADAMTS-4 and -5 immunoreactivity (IR) in striatum oriens (so), striatum radiatum (sr), striatum lacunosum moleculare (slm), and Dentate Gyrus molecular layer (DG ml). AU, arbitrary units, represent mean background-corrected pixel brightness measured on 4 sections per animal (n = 3 per genotype). *p<0.05, **p<0.01, statistics based on unpaired <i>t-test</i> with Welch's correction. (<b>E</b>) Optimized pepsin pre-treatment protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047793#pone.0047793-Doehner1" target="_blank">[50]</a> allowed the detection of ADAMTS-4 IR in extracellular protein depositions throughout the hippocampus of aged mice (lower and higher magnification). Scale bars: <b>A–C</b> = 500 µm; <b>E</b> = 200 µm.</p

ADAMTS-5 degrades Reelin.

<p>(<b>A–D</b>) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (<b>A</b>) Recombinant ADAMTS-5 (40 ng/µl) cleaves Reelin at both, its N- and C-cleavage site and further degrades the N-terminal fragment (asterisks). ADAMTS-5 activity is abolished after addition of TIMP-3 (20 ng/µl). (<b>B</b>) Pull-down (PD, using the G10 anti-Reelin antibody, diluted at 1:200, 1:100, 1:50) and subsequent IB with the same antibody revealed the existence of smaller N-terminal fragments (asterisks) in <i>wild-type</i> hippocampus homogenates. (<b>C</b>) Full length Reelin after 52 h incubation at 37°C (lane 1). ADAMTS-5 (80 ng/µl) was added to the FL-Reelin medium (indicated by +), which was previously incubated for 48 h with ADAMTS-4 (20 ng/µl, lane 2) or tPA (50 ng/µl, lane 4).Cleaved Reelin was incubated with ADAMTS-5 for additional 4 hours at 37°C (lanes 3 and 5). (<b>D</b>) Recombinant ADAMTS-1 (10, 20, 40 ng/µl) does not cleave Reelin. (<b>E</b>) Reelin enriched-medium was transferred to HEK293 cells expressing the indicated protease. The samples were collected 24 h after the medium transfer. (<b>F</b>) Schematic summary of Reelin processing and the possible modulations of this process. Dashed lines symbolize indirect effects of serpins and MMP-9 on Reelin processing, which are not mediated by tPA and ADAMTS-4, respectively. X = unknown ECM mediator.</p

Biochemical analysis of Reelin preoteolytic processing and protein levels of the identified Reelin proteases in young and old wild-type mice.

<p>Immunoblots using (<b>A</b>) anti-tPA (H-90), (<b>B</b>) anti-ADAMTS-4 (PA1-1749A), (<b>C</b>) anti-ADAMTS-5 (ab41037), (<b>D</b>) anti-Reelin (G10), (<b>E</b>) anti-Reelin (142), (<b>F</b>) anti-Actin (MAB1522) antibody. Lanes represent different animals. Hippocampus lysates from young (4 weeks) and old (16 months) animals were processed on the same gel and membrane. No difference in Reelin cleavage or levels of Reelin proteases is observed between young and old animals. However, a prominent Reelin-positive band of ∼60 kDa was selectively observed in the aged animals.</p

<i>w¹¹¹⁸</i> flies are not only optomotor blind but also dazzled by light.

<p>Courtship vigor indices were measured in single-choice courtship assays with mature males of indicated genotypes and receptive (<b>A</b>) or decapitated (<b>B</b>) <i>Ore-R</i> virgin females in daylight (yellow columns), under dim red light (red columns), or under low-intensity light conditions (orange columns). The numbers below the columns indicate the number of couples observed that initiated courtship, and error bars always represent double standard errors of the mean. Data for <i>ninaB^360d</i> males in panel A are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Krstic1" target="_blank">[4]</a>.</p

Effect of <i>white</i> mutation on sexual arousal of males.

<p>Models explaining possible effects of extra-retinal lack of White function on general sexual arousal of males. Increased sexual arousal of <i>white</i> males over time <i>t</i> may be caused by (<b>A</b>) a change in the general alertness from a ground state in wild-type flies (solid blue line) to an enhanced state of alertness in <i>w¹¹¹⁸</i> mutants (broken blue line), resulting in a shortened, sensitized period of latency to sustained courtship, <i>t_s</i>, as compared to the control period of latency, <i>t_c</i>, or (<b>B</b>) a reduction of the threshold from its wild-type (solid red line) to a reduced <i>w¹¹¹⁸</i> level (broken red line), which relevant stimuli need to reach to elicit sustained courtship, also resulting in a shortened, sensitized period of latency, <i>t_s</i>. Since the processing of various sensory information by the CNS is not affected in either model, both changes can be taken to result from an enhanced sensitivity of the CNS to courtship stimuli. (<b>C</b>) Alternatively, <i>w¹¹¹⁸</i> males may reach the threshold level for sustained courtship faster (broken green line in <i>w¹¹¹⁸</i> mutants with slope greater than that of solid green line in wild-type flies) because of an altered processing or integration of courtship information by the CNS or an enhanced sensitivity to relevant stimuli, for example pheromones, of sensory neurons in the PNS. Also in this model, the augmented sexual arousal leads to a shortened, sensitized period of latency to sustained courtship, <i>t_s</i>, because of a steeper slope of the broken versus the solid green line.</p

Chaining behavior of males is stimulated by a <i>w¹¹¹⁸</i> background.

<p>(<b>A</b>) In addition to the average chaining indices for groups of eight males <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Villella1" target="_blank">[30]</a> of indicated genotypes, the number of groups for which chaining was observed over the total number of groups tested is shown. Error bars represent double standard errors. The result for <i>Poxn-pRes</i> males is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Krstic1" target="_blank">[4]</a>. (<b>B</b>) Courtship chain of eight <i>w¹¹¹⁸</i>; <i>Poxn-pRes</i> males. The picture was taken 10 min after eight mature, but sexually naïve, <i>w¹¹¹⁸</i>; <i>Poxn-pRes</i> males were placed together into a small Petri dish.</p

MMP-9 activates Reelin-cleaving proteases.

<p>(<b>A–D</b>) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (<b>A</b>) Recombinant MMP-9 (10 ng/µl) induces Reelin cleavage. (<b>B</b>) Action of MMP-9 (20 ng/µl) on Reelin cleavage could be suppressed with TIMP-3 (10, 15, 20 ng/µl), α2M (10, 20, 40 ng/µl), and also with trypsin inhibitors SBTI and Apro (500 and 150, 1000 and 300, 2000 and 600 ng/µl). The framed lane belongs to the same IB (asterisk on the right blot), but was overexposed for visualization of cleaved Reelin. (<b>C</b>) Incubation of FL-Reelin and recombinant ADAMTS-4 (10 ng/µl), ADAMTS-5 (20 ng/µl), or MMP-9 (10 ng/µl) after heating the FL-Reelin medium at 80°C for 10 min. (<b>D</b>) Expression of the MMP-9 cDNA did not increase Reelin cleavage in Reelin expressing HEK293 cells (left), although we could confirm the synthesis of MMP-9 in these cells using anti-MMP-9 antibody (right). Short vertical lines at the bottom of the blots denote that the last lanes, from the same blot, were joined for visual presentation.</p

Lack of extra-retinal White function influences sexual orientation of males.

<p>Courtship vigor indices were measured in single-choice courtship assays with mature males of indicated genotypes and decapitated males (hatched columns) or decapitated females (filled columns) in the dark (red columns) or in daylight (yellow columns). The numbers below the columns indicate the number of males observed that initiated courtship. Data for <i>Ore-R</i> males are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077904#pone.0077904-Krstic1" target="_blank">[4]</a>.</p

Co-localization of ADMTS-4 and Reelin in the <i>striatum radiatum</i> of aged animals.

<p>(<b>A–D</b>) Double-immunofluorescence staining using anti-Reelin (red) and anti- ADAMTS-4 and anti-ADAMTS-5 (green) antibodies on brain sections of 15 month-old non-transgenic and 3xTg-AD mice. Co-localization analysis revealed a selective overlap of ADAMTS-4 IR (<b>A,B</b>), but not ADAMTS-5 IR (<b>C,D</b>), with Reelin in extracellular deposits in the striatum radiatum (sr). DAPI (blue) counterstaining was used for labeling the cell nuclei. Scale bars: 20 µm.</p