In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway

Background: Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. Previous studies indicate that mammalian MANF and CDNF support and protect brain dopaminergic system in non-cell-autonomous manner. However, MANF has also been shown to function intracellularly in the endoplasmic reticulum. To date, the knowledge on the interacting partners of MANF/CDNF and signaling pathways they activate is rudimentary. Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. Results: We first show that DmManf plays a role in the development of Drosophila wing. We exploited this function by using Drosophila UAS-RNAi lines and discovered novel genetic interactions of DmManf with genes known to function in the mitochondria. We also found evidence of an interaction between DmManf and the Drosophila homologue encoding Ku70, the closest structural homologue of SAP domain of mammalian MANF. Conclusions: In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies.Peer reviewe

Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes

Peer reviewe

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.Peer reviewe

Evoluutiossa konservoituneen hermokasvutekijän Manf karakterisointi banaanikärpäsmallissa

Among neurotrophic factors MANF/CDNF family is unique as their protein sequences are evolutionarily conserved between multicellular organisms. Still, little is known about their mechanism of action and interacting molecules. At the time of initiation of this study there were no known neurotrophic factors in invertebrates. According to the protein sequence homology there was an uncharacterized homologue to the novel neurotrophic factor MANF in Drosophila melanogaster. We found that Drosophila Manf (DmManf) is an essential gene in a fruit fly development. DmManf represents a true orthologue to mammalian MANF as its mutant lethality is rescued by human MANF. We have generated DmManf deletion mutant surviving to second instar larval stage with maternal contribution. When the maternal contribution of DmManf is abolished, the mutants die at the end of embryogenesis before hatching. In DmManf mutant the dopaminergic neurites degenerate and the dopamine level is extremely low. Ultrastructural analysis reveals nonapoptotic cell death in the embryonic ventral nerve cord and neuropile decomposition together with cell body glia activation taking place. In secretory cells like gastric caeca or fat body the visible loss of rough endoplasmic reticulum and drastic accumulation of vesicles, some filled with cellular debris, occur. According to microarray expression analysis data, expression of genes involved in vesicular transport and metabolism were altered in DmManf mutants. The expression of several genes implicated in pathology of Parkinson s disease (PD) was also altered. The degeneration of dopaminergic neurons is the hallmark for PD and this thesis work makes an effort to enlighten the mechanisms how the neurotrophic factor MANF protects these degenerating neurons.MANF/CDNF hermokasvutekijäperhe on ainutlaatuinen, koska sen aminohappojärjestys on evoluutiossa hyvin konservoitunut verrattuna toisiin hermokasvutekijäperheisiin. Tiedetään, että MANF ja CDNF pystyvät suojaamaan dopamiinivälitteisiä hermosoluja rotan Parkinsonin taudin mallissa. Dopamiinivälitteisten hermosolujen tuhoutuminen on ominaista Parkinsonin taudille ja siihen ei ole toistaiseksi olemassa parantavaa lääkitystä. Kuitenkin MANF/CDNF-proteiinien tarkka toimintamalli ja yhteistyökumppanit ovat toistaiseksi tuntemattomia. Tämän tutkimuksen alkuvaiheessa selkärangattomilla eläimillä ei tunnettu ainuttakaan hermokasvutekijää. MANF:n proteiinijärjestyksen homologian perusteella löydettiin MANF:n banaanikärpäsvastine, joka osoittautui aiemmin tutkimattomaksi geeniksi Drosophilassa. Banaanikärpäsen Manf:n homologia tutkimalla pyrimme selvittämään yleispäteviä toimintamalleja kyseiselle hermokasvutekijäperheelle. Hyvä esimerkki MANF-perheen korkeasta konservoitumisasteesta evoluutiossa myös toiminnan osalta on koe, jossa pelastetaan Drosophila Manf poistogeenisen banaanikärpäsen elinkyky ihmisen MANF-proteiinia tuottavan siirtogeenin avulla. Tutkimuksissani ilmeni, että Manf on oleellinen myös Drosophilalla dopamiinivälitteisten hermosolujen selviytymisessä ja Manf:n poistogeenisen banaanikärpästoukan dopamiinivälitteiset hermosolut surkastuvat. Vertailemalla muutoksia koko genomin geeniekspression tasolla Manf:n poisto- ja siirtogeenisten banaanikärpästen välillä löysimme joukon Manf:n toiminnalle ominaisia prosesseja sekä ehdokasgeenejä, joita on mahdollista tutkia jatkossa sekä banaanikärpäs- että nisäkäsmalleissa

Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless

Pitt-Hopkins syndrome (PTHS) is caused by haploinsufficiency of Transcription factor 4 (TCF4), one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da), homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder

Gene expression analysis of <it>Drosophilaa Manf </it>mutants reveals perturbations in membrane traffic and major metabolic changes

Abstract Background MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. Results The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. Conclusions We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.</p

El Diario de Pontevedra : periódico liberal: Ano XXXIII Número 9565 - 1916 marzo 9

Results from ubiquitous knockdown studies of UAS-RNAi lines with and without DmManf overexpression. A pdf file. (PDF 10 kb

Drug-induced ER stress upregulates <i>DmManf</i> expression.

<p>A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of <i>DmManf</i> and <i>Hsc3</i> were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of <i>Xbp1</i>, unspliced (<i>Xbp1</i>^<i>u</i>) and spliced (<i>Xbp1</i>^<i>s</i>). <i>RpL32</i> was used as a loading control. C–D) qPCR analysis of <i>Hsc3</i> and <i>Xbp1</i> expression in <i>DmManf</i> mutant (C) and <i>DmManf</i> overexpressing (D) larvae. Expression of <i>Hsc3</i> was not altered but <i>Xbp1s</i> mRNA level was increased in response to overexpression of <i>DmManf</i>. The overexpression of <i>DmManf</i> resulted in 165-fold increase in <i>DmManf</i> mRNA level (±23, P<0.001, not shown). <i>Xbp1t</i>, total amount of <i>Xbp1</i>; <i>Xbp1s</i>, spliced-specific transcript of <i>Xbp1</i>; <i>Xbp1 s</i>:<i>t</i>, proportion of <i>Xbp1s</i> out of <i>Xbp1t</i>. OE, overexpression. Average ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001 versus control, Student’s t-test.</p

Overview of genetic interactions between <i>DmManf</i> and selected ER- and UPR-related genes.

<p>A) UAS-RNAi lines were crossed to MS1096-GAL4 and 69B-GAL4 driver lines in wild type and <i>DmManf</i>-overexpressing backgrounds. The observed phenotypes of knockdown flies in <i>DmManf</i>-overexpressing background (OE vs. wt) were compared to the phenotype of knockdown flies in wild type background. Yellow (stronger phenotype) represents affected phenotypes. Light gray (no phenotype in either background), gray (phenotype similar in both backgrounds) and dark gray (lethal phenotype in both backgrounds) represent cases where the overexpression of <i>DmManf</i> did not affect the phenotype caused by knockdown of target gene. As a comparison, results from our previously published microarray analysis (MAA) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151550#pone.0151550.ref020" target="_blank">20</a>] are presented; red and blue indicate up- and down-regulation of the target gene, respectively. Mutant larvae stands for zygotic <i>DmManf</i>^<i>Δ96</i> mutant larvae, OE larvae for 69B-GAL4>UAS-<i>DmManf</i>^L3 larvae, and mutant embryos for maternal and zygotic <i>DmManf</i>^{<i>Δ96mz</i>} mutant embryos. B) Overexpression of DmManf by UAS-DmManf^L5 with either semi-ubiquitous 69B-GAL4 or with wing driver MS1096-GAL4 did not result in any obvious phenotypes in adult flies. In MS1096-GAL4 line, we detected a weak GAL4 expression in CNS as well presenting a probable reason for the lethal phenotypes we observed in our knockdown experiments. However, for screening we only monitored the adult wing phenotype. ER, endoplasmic reticulum; ERAD, ER associated degradation; ERSS, ER stress sensor protein; MAA, microarray analysis; OE, overexpression.</p

<i>Xbp1</i> is a genetic interactor of <i>DmManf</i>.

<p>A–B) The knockdown of <i>Xbp1</i> with wing driver MS1096-GAL4 (A) and semi-ubiquitous 69B-GAL4 driver (B) resulted in stronger phenotype when <i>DmManf</i> was simultaneously overexpressed (+ <i>DmManf</i> OE) in comparison to wild type background. Simultaneous expression of UAS-mCD8-GFP (+ mGFP) did not affect the <i>Xbp1</i> knockdown phenotypes. Scale bar 1 mm. C) Quantitative RT-PCR analysis of knockdown of <i>Xbp1</i> with ubiquitous <i>tub</i>-GAL4 driver in 50–54 h AEL larvae. Knockdown of <i>Xbp1</i> showed minimal decrease in <i>DmManf</i> mRNA level and small decrease in <i>Hsc3</i> levels. The amounts of both total and spliced <i>Xbp1</i> transcripts were clearly decreased indicating a successful knockdown of <i>Xbp1</i> gene. KD, knockdown; OE, overexpression. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151550#pone.0151550.s002" target="_blank">S2 Fig</a> for further data. Average ± standard deviation.</p