Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators

The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD

Sensory Integration Regulating Male Courtship Behavior in Drosophila

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior

Influence of the White locus on the courtship behavior of Drosophila males.

Since its discovery by Morgan, the Drosophila white gene has become one of the most intensely studied genes and has been widely used as a genetic marker. Earlier reports that over- and misexpression of White protein in Drosophila males leads to male-male courtship implicated white in courtship control. While previous studies suggested that it is the mislocalization of White protein within cells that causes the courtship phenotype, we demonstrate here that also the lack of extra-retinal White can cause very similar behavioral changes. Moreover, we provide evidence that the lack of White function increases the sexual arousal of males in general, of which the enhanced male-male courtship might be an indirect effect. We further show that white mutant flies are not only optomotor blind but also dazzled by the over-flow of light in daylight. Implications of these findings for the proper interpretation of behavioral studies with white mutant flies are discussed

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

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

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

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