Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis

Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency

CITATION: Di Meo, I., et al. 2017. Acetyl-4′-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency. Scientific Reports, 7:11260, doi:10.1038/s41598-017-11564-8.The original publication is available at https://www.nature.comCoenzyme A is an essential metabolite known for its central role in over one hundred cellular metabolic reactions. In cells, Coenzyme A is synthesized de novo in five enzymatic steps with vitamin B5 as the starting metabolite, phosphorylated by pantothenate kinase. Mutations in the pantothenate kinase 2 gene cause a severe form of neurodegeneration for which no treatment is available. One therapeutic strategy is to generate Coenzyme A precursors downstream of the defective step in the pathway. Here we describe the synthesis, characteristics and in vivo rescue potential of the acetyl-Coenzyme A precursor S-acetyl-4′-phosphopantetheine as a possible treatment for neurodegeneration associated with pantothenate kinase deficiency.https://www.nature.com/articles/s41598-017-11564-8Publisher's versio

Wnt5 protein and mRNA expression domains in epidermis, muscle and tendon cells during embryonic development.

<p>WNT5 is predominantly expressed in subsets of neurons in the CNS from stage 12 onwards throughout embryonic development (data not shown; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone.0032297-Fradkin2" target="_blank">[28]</a>). However, there is also strong expression from this stage onwards in the epidermis and the musculature. At stage 12, Wnt5 protein (<b>A, B</b>) and <i>Wnt5</i> mRNA (<b>E, F</b>) expression is observed in the epidermis, most prominently in two clusters (<b>arrows</b>), and throughout the somatic mesoderm that will give rise to the body wall musculature. Later in embryonic development at early stage 16 WNT5 protein and <i>Wnt5</i> mRNA are present in the attachment sites (<b>arrows in panels C and G</b>) and at low levels in most muscle fibers including the LTMs 21, 22 and 23 (<b>C, G</b>). At the end of embryonic development at late stage 17, Wnt5 protein (<b>D</b>) and <i>Wnt5</i> mRNA (<b>H</b>) are almost undetectable in the somatic mesoderm. In all panels anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants frequently do not express SR, while the bypassed attachment sites do.

<p>Double labeled stage 16 embryos are shown of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) with anti-Muscle Myosin in green and anti-SR in red (Material and Methods). Asterisks mark the novel attachment sites of the overshooting LTM muscles; white arrowheads mark the locations of the original attachment sites. In <i>Wnt5</i> mutants the novel target sites do not express SR in 65% of the segments containing overshooting muscles, while the bypassed attachment sites usually express SR. The SR positive, original tendon cell is also present in <i>drl^Red2</i> mutants, but is partly masked by the overshooting muscle fiber in panel (<b>C</b>), but clearly visible in panel (F)). These results were confirmed in embryos that express Tau-MYC under the control of a <i>stripe</i> promoter in both <i>Wnt5</i> and <i>drl</i> mutants (data not shown). The following genotypes are shown, the control UAS-Tau-MYC; <i>sr</i>-GAL4 embryos (<b>D</b>), <i>Wnt5⁴⁰⁰</i>; UAS-Tau-MYC/<i>sr</i>-GAL4 (<b>E</b>) and <i>drl^Red2</i>; UAS-Tau-MYC/<i>sr</i>-Gal4 (<b>F</b>). Anti-Muscle Myosin is shown in green and anti-MYC in red. No MYC protein is observed in the ectopic attachment sites. The photographs in Panels (<b>A</b>–<b>C</b>) were taken on a compound microscope and those in Panels (<b>D–F</b>) on a confocal microscope. Anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants express βPS integrin.

<p>Wild type (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^red2</i> (<b>C</b>) embryos were labelled with anti-βPS Integrin. Muscles 21–23 do exhibit an accumulation of βPS Integrin protein at the tip of the overshooting fibers (white asterix).</p

Muscle attachment defects persist from the embryonic to larval stages in <i>Wnt5</i> and <i>drl</i> mutants.

<p>Third instar larval body walls of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) mutant larvae are stained with anti-FAS2 (mAb 1D4). <i>Wnt5⁴⁰⁰</i> larvae and <i>drl^Red2</i> larvae frequently bypass their normal attachment sites and extend ventrally where they form new stable attachments. The original and ectopic tendons cells are indicated by + and *, respectively. FAS2 protein is evident at both sites. The penetrance of the bypass phenotypes is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. Anterior is up and ventral is left.</p

LTM muscle fibers 21, 22 and 23 frequently overshoot their attachment sites in <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutant embryos.

<p>Stage 16 embryo body wall muscle preparations stained with anti-Muscle Myosin are shown for the wild type control (<i>w¹¹¹⁸</i>) (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>), <i>drl^Red2</i> (<b>C</b>), <i>Drl-2^E124</i> (<b>D</b>), <i>dnt^42.3</i> (<b>E</b>) and Df(2L)Exel6043 (<b>F</b>). Two hemisegments are displayed for each genotype with one set of muscles 21–23 labelled. In <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutants, LTMs frequently bypass their normal attachment at the epidermis at muscle 12 and instead extend ventrally beyond muscle 13 and attach at a novel epidermal site located close to muscle fiber 7. Df(2L)Exel6043 mutant embryos, that lack both DNT and DRL, display this phenotype in all hemisegments of the homozygous animals. The penetrance of these phenotypes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. The muscle bypass phenotype is schematically shown in panel (<b>G</b>). The * indicates the location of the novel, ectopic epidermal attachment in panels (<b>B</b>), (<b>C</b>), (<b>E</b>), (<b>F</b>) and (<b>G</b>). Anterior is up and ventral is left.</p

Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors

In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB) axons, whose α and β branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed), which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL’s ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons

Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

The VPS13A gene is associated with the neurodegenerative disorder Chorea Acanthocytosis. It is unknown what the consequences are of impaired function of VPS13A at the subcellular level. We demonstrate that VPS13A is a peripheral membrane protein, associated with mitochondria, the endoplasmic reticulum and lipid droplets. VPS13A is localized at sites where the endoplasmic reticulum and mitochondria are in close contact. VPS13A interacts with the ER residing protein VAP-A via its FFAT domain. Interaction with mitochondria is mediated via its C-terminal domain. In VPS13A-depleted cells, ER-mitochondria contact sites are decreased, mitochondria are fragmented and mitophagy is decreased. VPS13A also localizes to lipid droplets and affects lipid droplet motility. In VPS13A-depleted mammalian cells lipid droplet numbers are increased. Our data, together with recently published data from others, indicate that VPS13A is required for establishing membrane contact sites between various organelles to enable lipid transfer required for mitochondria and lipid droplet related processes

Impaired Vps13 function leads to defects in protein homeostasis.

<p>(A) Percentage of isogenic control and <i>Vps13</i> mutant flies that eclosed at increasing temperatures. (B) Percentage of homozygous <i>Vps13</i> mutant flies and excision line flies that eclosed at 29°C. (C) Percentage of flies of various genotypes that eclosed at 29°C. Two independent deficiency lines (lacking a genomic area containing the <i>Vps13</i> gene) were crossed with <i>Vps13/ CyO</i> heterozygous flies. Eclosion rate of the following genotypes was analyzed: <i>Vps13/+</i>, <i>Df #7535/+</i>, <i>Vps13/Df #7535</i>, <i>Df #7534/+</i> and <i>Vps13/Df #7534</i>. (D) Percentage of <i>Vps13</i> flies that eclosed at 22°C on food with increasing concentrations of L-canavanine. (E) Western blot analysis of lysates of 1 day old control and <i>Vps13</i> mutant fly heads. Ubiquitylated proteins, K48 ubiquitylated proteins and K63 ubiquitylated proteins were detected. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p