Retromer deficiency in Tauopathy models enhances the truncation and toxicity of Tau.

Alteration of the levels, localization or post-translational processing of the microtubule associated protein Tau is associated with many neurodegenerative disorders. Here we develop adult-onset models for human Tau (hTau) toxicity in Drosophila that enable age-dependent quantitative measurement of central nervous system synapse loss and axonal degeneration, in addition to effects upon lifespan, to facilitate evaluation of factors that may contribute to Tau-dependent neurodegeneration. Using these models, we interrogate the interaction of hTau with the retromer complex, an evolutionarily conserved cargo-sorting protein assembly, whose reduced activity has been associated with both Parkinson’s and late onset Alzheimer’s disease. We reveal that reduction of retromer activity induces a potent enhancement of hTau toxicity upon synapse loss, axon retraction and lifespan through a specific increase in the production of a C-terminal truncated isoform of hTau. Our data establish a molecular and subcellular mechanism necessary and sufficient for the depletion of retromer activity to exacerbate Tau-dependent neurodegeneration.post-print2287 K

Examination of transcriptional regulation and functional roles of axon guidance receptors in Drosophila dorsal vessel formation

THESIS 10412Early steps of cardiogenesis in vertebrates and invertebrates are similar in that in both systems, bilateral rows of cardiac progenitors migrate towards a point where the two rows meet and make a tubular structure. As with any other developmental processes, proper cardiac migration requires proper guidance of progenitors towards the target position. Drosophila dorsal vessel (DV; Drosophila equivalent of heart) progenitors have been shown to express a number of surface molecules found to be important for axon projection, implying that migration of these cells also requires guidance

Tinman Regulates NetrinB in the Cardioblasts of the Drosophila Dorsal Vessel.

Morphogenesis of the Drosophila dorsal vessel (DV) shares similarities with that of the vertebrate heart. Precursors line up at both sides of the embryo, migrate towards the midline and fuse to form a tubular structure. Guidance receptors and their ligands have been implicated in this process in vertebrates and invertebrates, as have been a series of evolutionarily conserved cardiogenic transcriptional regulators including Tinman, the Drosophila homolog of the transcription factor Nkx-2.5. NetrinB (NetB), a repulsive ligand for the Unc-5 receptor is required to preserve the dorsal vessel hollow. It localizes to the luminal space of the dorsal vessel but its source and its regulation is unknown. Here, using genetics together with in situ hybridization with single cell resolution, we show how tin is required for NetrinB expression in cardioblasts during DV tubulogenesis and sufficient to promote NetB transcription ectopically. We further identify a dorsal vessel-specific NetB enhancer and show that it is also regulated by tin in a similar fashion to NetB

NetB protein expression, in <i>tin</i> mutants is downregulated in CBs.

<p>As <i>NetB</i> mRNA is almost absent in CBs of <i>tin-ABD</i>; <i>tin</i>^<i>346</i>/<i>tin</i>^<i>346</i> mutants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.g003" target="_blank">Fig 3</a>), we tested the NetB-tm protein expression in this background using the transmembrane V5-tagged NetB knock-in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref018" target="_blank">18</a>]. A and A’ represent NetB expression in <i>tinABD; tin</i>^<i>346</i>/<i>+</i> heterozygous embryos (full confocal stack in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.s001" target="_blank">S1 Fig</a>). There is no significant difference in the NetB-tm levels between <i>wild-type</i> and heterozygotes. However, in <i>tin-ABD</i>; <i>tin</i>^<i>346</i>/<i>tin</i>^<i>346</i>mutants, NetB-tm expression level is dramatically reduced (B, B’, full confocal stack in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.s002" target="_blank">S2 Fig</a>) while its expression in the muscles [M] is unaffected (C). CBs are labeled with a-Mef2 (Magenta) and NetB is labeled with a-V5 (green). All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel (^***p<2.6x10^-5).</p

The <i>NetB</i>-M reporter is expressed in CBs and weakly in muscles.

<p>(A) Schematic representation of the positions and the relative sizes of the available <i>Gal4</i> reporters for <i>NetB</i> locus. We screened Gal4 from the Janelia collection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref022" target="_blank">22</a>] for DV expression. The line expressing Gal4 efficiently in the DV is shown in magenta. (B-C’) Gal4 expression pattern was examined using a UAS-<i>Tau-Myc-GFP</i> construct. Co-staining of embryos with a-Myc (green) and a-Mef2 (magenta) revealed Gal4 expression in muscles and the DV at stage 13 (B, B’) and 15 (C, C’). Co-staining of these embryos with a-Myc (green), a-Mef2 (red) and a-Zfh1 (magenta) reveals a reduction in the expression in other in muscles and pericardial cells while expression in CBs persists at late stage 16–17 (D, D’). All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel.</p

<i>tin</i> regulates <i>NetB</i> mRNA expression <i>in vivo</i>.

<p>A and A’ represent <i>NetB</i> mRNA (magenta) expression in CBs (green), labeled with Tau-Myc driven by <i>TinC-Gal4</i> to label CBs, in heterozygous <i>tinABD</i>; <i>tin346</i>/<i>+</i> embryos (arrowheads). <i>NetB</i> expression dramatically drops in homozygous <i>tinABD</i>;<i>tin346</i>/<i>tin346</i> late stage 16 embryos (B and B’; open arrowhead-asterisks in B’ point to where CBs are positioned in B). All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel. C, quantification of the in situ signal in cardioblast from the anterior, aorta region (A) or the posterior, heart proper (P) region of the dorsal vessel compared to transversal epidermal signal (Ep) in <i>tinABD</i>; <i>tin346</i>/<i>+</i> and <i>tinABD</i>;<i>tin346</i>/<i>tin346</i> embryos (^***p<0.003 and ^**p<0.01).</p

Ectopic expression of Tin induces NetB ectopic expressions <i>in vivo</i>.

<p>In a wild-type background, staining for NetB protein (magenta), displays little or no NetB expression in the Engrailed stripes (A and A’). However, in an <i>engrailed-Gal4</i>>UAS-<i>tin</i> background, NetB is strongly induced in Engrailed stripes which now ectopically express Tin (B and B’, arrowheads). For NetB protein staining, the transmembrane V5-tagged NetB-tm knock-in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref018" target="_blank">18</a>] was used. All panels are lateral views with anterior to the left and dorsal side up. A magnification of the regions delineated by insets is shown for each panel.</p

Tin binds directly to NetB-M enhancer element.

<p>(A) ChIP analysis of the <i>NetB-M</i> genomic loci in S2R+ cells transfected with p<i>Act5C-GFP-tinman</i>. The precipitated DNA fragments were amplified by real-time qPCR using overlapping primers (R1-R20) designed over the genomic region covering the <i>NetB-M</i> enhancer element. ChIP signal is also illustrated as a curve peaking at R1, R10/R11, R14 and R16. A schematic of <i>NetB</i> locus is also illustrated below the graph. Note that the schematic view represents only a ~2kb region (chrX:14,744,635–14,746,673) of the <i>NetB-M</i> enhancer that showed significant enrichment as no significant enrichment for the rest of the enhancer was detected above the background. Enrichment is presented as a percentage of total input. (B) Consensus Tin-binding motifs detected with JASPAR within the ChIP enriched region and their conservation in different <i>Drosophila</i> species (<i>mel</i>, <i>melanogaster; sim</i>, <i>simulans; sec</i>, <i>sechellia; yak</i>, <i>yakuba; ere</i>, <i>erecta</i>).</p

Schematic representation of the <i>Drosophila</i> dorsal vessel’s cellular composition and organization.

<p>Top, presents <i>Drosophila</i> DV at embryonic stage 15 while cardiac progenitors are migrating towards the dorsal midline. Bottom is an schematic representation of the embryonic heart proper at stage 17 when the DV lumen has formed. Aortic portion (right) is oriented anteriorly and the beating (heart) portion (left), posteriorly. Based on the expressed marker TFs, different cell types are colored. CBs are divided into Tin-(green) or Svp-expressing SMCs (light green) subtypes. SMCs of the last three posterior segments of DV make the future ostial cells (inflow valves). CBs are surrounded by pericardial cells (PCs; blue) on their ventrolateral side. PCs are divided into Tin-positive (blue) or Tin-negative PCs (not pictured). Bottom right, is a schematic representation of cross-section view of the DV lumen at stage 17. CBs on the opposite sides assume a crescent-like shape by contacting each other at the two dorsal and ventral apical sides, avoiding contact at the luminal domains, therefore making a hollow space in between. Unc-5 and NetB have been proposed to play a role in the formation of the hollow space between each CB on each row and its counterpart on the opposite row [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref009" target="_blank">9</a>].</p

NetB is expressed by CBs and can be detected in the luminal space after dorsal vessel closure.

<p>(A and B) Myc staining of embryos expressing a secreted Myc-tagged NetB knock-in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref018" target="_blank">18</a>] (magenta, luminal space) reveals secretion of NetB into the luminal space. CBs and PCs are labeled with a-Mef2 (green) and a-Zfh1 (cyan), respectively. Two cross sections taken from two points numbered 1 and 2 (places of which indicated in A and B) are shown on the right. The cross section at the point 1 is depicted schematically on the right and also in E. Note that due to secretion from CBs, NetB-Myc is mainly concentrated in the luminal space. (C and D) Co-staining for Tin (magenta) and a transmembrane V5-tagged NetB knock-in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148526#pone.0148526.ref018" target="_blank">18</a>] (green) reveals NetB expression in Tin-positive cardioblasts. Note the regular gaps in NetB-tm expression corresponding to Tin-negative SMCs (arrowheads). All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel in A and B.</p