Untersuchungen zur biologischen Funktion der kleinen GTPase Centaurin gamma in <em>Drosophila melanogaster</em>

Bewegungsmangel und ein Überangebot an kalorienreicher Nahrung haben dazu geführt, dass die globale Ausbreitung von metabolischen Erkrankungen wie Adipositas und Typ-2-Diabetes (T2D) in westlichen Kulturgesellschaften die Dimension einer Epidemie erreicht hat. Die Aufklärung der molekularen Mechanismen von Stoffwechselfunktionen wie Energiespeicherung und -mobilisierung ist von zentraler Bedeutung für das Verständnis der Ätiologie dieser Erkrankungen. Die Fruchtfliege Drosophila melanogaster ist ein ausgezeichnetes Modellsystem zur Entschlüsselung der molekularen Grundlagen von Stoffwechselprozessen. Viele grundlegende Stoffwechselfunktionen sind zwischen Vertebraten und Drosophila konserviert. Fliegen sind in der Lage ihre Menge an zirkulierenden Zuckern in der Hämolymphe zu modellieren und sie speichern überschüssige Nährstoffreserven in Form von Glykogen und Lipiden, welche sie bei Bedarf mobilisieren können. Durch eine kalorienreiche Diät können auch in Drosophila Phänotypen induziert werden, die Ähnlichkeit zu Adipositas und T2D aufweisen. Adipositas und T2D sind im Menschen mit einer Fehlregulation des Insulin-Signalweges assoziiert. Die Insulin-Signalkaskade ist im Tierreich hoch konserviert und spielt unter anderem eine essentielle Rolle bei der Regulation des Glukose-Stoffwechsels und bei Wachstums- und Größenkontrolle. Das Drosophila ArfGEF Steppke, ein Mitglied der Cytohesin-Familie von Guanin-Nukleotid-Austauschfaktoren, konnte vor einigen Jahren als Schlüsselkomponente des Insulin-Signalwegs identifiziert werden. ArfGEF Proteine vermitteln die Ablösung von gebundenem GTP an ArfGTPasen, die wiederum intrazelluläre Transportprozesse und die Struktur von Organellen kontrollieren. Gegenspieler der ArfGEF Proteine sind die ArfGAP Proteine, die die Hydrolyse von Arf-gebundenem GTP katalysieren. In einem Zellkultur-basierten RNAi Screen konnte in Vorarbeiten centaurin gamma (centg) als putatives ArfGAP im Insulinsignalweg von Drosophila identifiziert werden. Centaurine bilden eine Familie von Multidomänenproteinen und haben sowohl in Drosophila als auch in Säugern die gleiche, einzigartige Domänenstruktur. Mitglieder der Centg Proteinfamilie besitzen eine GTPase Domäne, eine PH Domäne, eine ArfGAP Domäne und Ankyrin Wiederholungsmotive. Im Rahmen dieser Arbeit sollte untersucht werden, ob das Drosophila centg Gen in vivo als Regulator des Insulin-Signalweges fungiert. Durch biochemische Untersuchungen konnte im ersten Teil der Arbeit nachgewiesen werden, dass Centg für eine funktionelle GTPase kodiert, die durch ihre interne GAP Domäne katalysiert wird. Zur Analyse der in vivo Funktion wurde mittels ends-out gene targeting eine knockout Mutante für centg generiert. centg-/- mutante Tiere sind homozygot lebensfähig. Sie zeigen jedoch im Vergleich zu anderen Regulatoren des Insulinsignalweges keinen Wachstumsphänotyp in den einzelnen Entwicklungsstadien und auch keine veränderte 4E-BP Expression. Außerdem weisen sie keine veränderte Sensitivität gegenüber Hungerbedingungen und keine Einschränkungen bei der Mobilisierung der Speicherlipide auf. Weiterhin zeigten Fütterungsexperimente mit hochkalorischem Futter, dass sich centg-/- Mutanten unter hochkalorischen Bedingungen stark verzögert entwickeln und exzessiv Speicherlipide akkumulieren, ähnlich wie dies bei T2D bzw. Adipositas vorkommt. Im Gegensatz dazu sind Centg1 knockout Mutanten der Maus, die mit hochkalorischem Futter gefüttert wurden, gegen ernährungsbedingte Adipositas und Insulinresistenz geschützt. Wenn das Drosophila centg-Gen nicht am Insulin-Signalweg beteiligt ist, welche Rolle erfüllt es dann? Expressionsstudien ergaben erste Hinweise darauf, dass centg eine wichtige Funktion im Nervensystem übernimmt. Imaging Analysen zeigen, dass centg-/- Mutanten bzw. Larven, die centg in den Körperwandmuskeln überexprimieren, Defekte an Motorneuronen beziehungsweise an Körperwandmuskeln aufweisen. In der Mutante innervieren 25 Prozent der Synapsen ihren Muskeln entweder an der falschen Stelle oder fehlen ganz. Außerdem ist die Morphologie von 25 Prozent der untersuchten Muskeln verändert. Die Überexpression in den Muskeln führt zu noch stärkeren Defekten. In 37% der Fälle führt die Überexpression zu Muskeldefekten und in 11% hat die Überexpression einen Einfluss auf die Synapse. Centg scheint somit eine Funktion in Motorneuronen und deren korrekter Interaktion mit den Muskeln zu übernehmen. Die generierten genetischen und biochemischen Werkzeuge werden es in der Zukunft ermöglichen, weitere Funktionen von centg z.B. bei Insulin-abhängigen Wachstumsprozessen im ZNS oder im Glutamatrezeptor-Signalweg zu untersuchen

The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila

Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar larval transition

<i>Drosophila</i> Ceng1A is a functional GTPase with a catalytic internal GAP domain.

<p>(A) Schematic representation of the predicted domain structure of the three murine PIKE isoforms (PIKE-A, -L and –S) and the homologous <i>Drosophila</i> Ceng1A isoforms (Ceng1A-PA, -PB and -PC). <i>ceng1A</i> codes for a N-terminal GTPase domain, a PH domain, a GAP domain and an ankyrine motif. Predicted domains of the three <i>Drosophila</i> Ceng1A proteins with PIKE domains show a high degree of conservation. Numbers indicate percentage of identity [similarity] on the amino acid level. (B) The GTPase and GAP domains of Ceng1A were used for a colorimetric in vitro GTPase assay. Two constructs were cloned and expressed in <i>E. coli</i> for the analysis: A 6xHis tagged construct containing the C-terminus of Ceng1A including the GAP domain and a 6xHis tagged construct containing the GTPase domain. (B') The graph depicts absorption at 635 nm versus protein concentration in µM. Addition of the GAP domain increases GTP hydrolysis of the GTPase domain. (B’’) Relative amount of hydrolyzed GTP. The GTPase domain alone shows modest GTPase activity, but activity was increased 1.5-fold by including the GAP domain. (C) Gene locus organization and generation of <i>ceng1A</i> knock-out mutants. Exon/intron structure of the <i>ceng1A</i> locus is depicted. Start sites of the three transcripts (<i>ceng1A</i> -RA, -RB and –RC) are indicated. A loss-of-function mutation for <i>ceng1A</i> was generated by ends-out gene targeting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097332#pone.0097332-Huang1" target="_blank">[19]</a>. The targeting construct for the homologous recombination was designed to delete exons 5–10, which encode all functional domains. (C') <i>Real-time</i> RT-PCR analysis of <i>ceng1A</i> expression in the generated <i>ceng1A</i> mutants.</p

<i>real-time</i> RT-PCR primers.

<p><i>real-time</i> RT-PCR primers.</p

<i>ceng1A</i> mutants show delayed second instar larval stage and reduced ecdysone signaling.

<p>Relative amount of control (A) and <i>ceng1A</i> mutants (B) in L1, L2, L3 and pupal stage was determined from egg lay (0) to 140 hours after egg deposition. n = 3; each n corresponds to 50 larvae; error bars indicate SEM. <i>ceng1A</i> mutants are delayed in second instar larval stage compared to controls. (C – E) Onset of pupariation <i>ceng1A</i> mutant or wildtypic larvae on standard food (C, NFD), high fat diet (D, HFD) and high sugar diet (E, HSD) was analyzed. Delay in development of <i>ceng1A</i> mutant larvae is nutrition independent. n = 3; each n equals 50 larvae; error bars indicate SEM (F) Scheme of average stage length in control and <i>ceng1A</i> mutant larvae (derived from A and B). Growth rate was assessed as an increase of weight over time from egg deposition to pupariation. The main delay of growth is between 45 and 80 hours after egg lay (corresponding to second instar larval stage). Afterwards, growth rate of <i>ceng1A</i> mutants increases in parallel to the control growth rate, only with the in L2 stage accumulated time delay. (G) From 48 hours after egg deposition to pupariation, expression of the ecdysone target genes <i>E75B</i> was analyzed via <i>real-time</i> RT-PCR. Peaks of <i>E75B</i> expression coincide in control and <i>ceng1A</i> mutant animals. However, <i>E75B</i> induction levels are reduced in <i>ceng1A</i> mutants. Transparent yellow bars correlate peaks of <i>E75B</i> expression with the growth rate at that time point. n = 5 for all experiments; error bars indicate SEM.</p

Figure 3

<p>. <b>Ceng1A is not involved in metabolic control.</b> (A) <i>ceng1A</i> mutant larvae feed normally, since quantitative analysis of Brilliant Blue FCF-colored yeast uptake shows no difference between control and <i>ceng1A</i> mutants. n = 10, each n equals 10 larvae; error bars indicate SEM. <i>Real-time</i> RT-PCR analysis of early third instar larvae (B) and dissected CNS (C) shows that transcription of <i>InR</i>, <i>4EBP</i>, <i>lip3, dilp2, 3</i> and <i>5</i> as well as <i>PTTH</i> is not changed in <i>ceng1A</i> mutants. n = 10; error bars indicate SEM; n.s.  = p>0,5 (t-test). (D) Immunoblots using anti-pAMPK and anti-Akt antibodies indicate that there is no difference in AMPK and Akt phosphorylation between control and <i>ceng1A</i> mutant larvae in starvation and fed conditions. Anti-actin served as a loading control. Quantification (D') of western blots of control and <i>ceng1A</i> mutant larvae stained for pAMPK relative to loading control. n = 3; error bars indicate SEM. (E) Survival curve of <i>ceng1A</i> mutant and <i>w-</i> control flies under starvation conditions. The mean survival time of <i>ceng1A</i> mutants (53,6 h) is not significantly changed compared to <i>w-</i> control flies (50,5h). p>0.5 (log rank test) (F) Thin layer chromatography to determine triacylglyceride (TAG) levels of control and <i>ceng1A</i> mutant flies upon starvation. Height of TAGs is shown in the right lane. Samples were taken at the start of the experiment (0h) and after 20 and 28 hours of starvation. (F') Body TAG levels are not changed in <i>ceng1A</i> mutants. Graph represents the relative absorbance of TAG bands quantified by photo densitometry. n = 3; error bars indicate SEM (G) Oil Red O staining of third instar fat bodies. Larvae were grown on either standard food (NFD) or high-sugar food (HSD). Fat body storage lipid load of <i>ceng1A</i> mutants does not differ from control larvae. (G') Analysis of relative lipid droplet size reveals no differences between control and <i>ceng1A</i> mutant larvae under both conditions. n = 50; error bars indicate SEM; p>0,5 (t-test).</p

<i>ceng1A</i> mutants do not differ from control animals in size and weight in larval, pupal or adult stages.

<p>For all experiments zygotic and maternal <i>ceng1A</i> mutant animals were used. (A – A’’) Comparison of <i>ceng1A</i> mutant adults, larvae and pupa with wildtypic counterparts does not reveal any morphological defects. (B) Length of third instar <i>ceng1A</i> mutant pupae is not altered relative to controls. (C) Total weight (in mg) as well as area of the wings (D) does not differ in adult <i>ceng1A</i> mutants compared to controls. n = 50; error bars indicate SEM; p>0,5 (t-test).</p

Generation of a homozygous GBA deletion human embryonic stem cell line

We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase) protein expression

Path mediation analysis reveals GBA impacts Lewy body disease status by increasing α-synuclein levels

Synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal α-synuclein in intraneuronal inclusions, named Lewy bodies. Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase, have been identified as the most common genetic risk factor for PD and DLB. However, despite extensive research, the mechanism by which glucocerebrosidase dysfunction increases the risk for PD or DLB still remains elusive. In our study we expand the toolbox for PD-DLB post-mortem studies by introducing new quantitative biochemical assays for glucocerebrosidase and α-synuclein. Applying causal modelling, we determine how these parameters are interrelated and ultimately impact disease manifestation. We developed quantitative immuno-based assays for glucocerebrosidase and α-synuclein (total and phosphorylated at Serine 129) protein levels, as well as a liquid chromatography–mass spectrometry method for the detection of the glucocerebrosidase lipid substrate glucosylsphingosine. These assays were applied on tissue samples from frontal cortex, putamen and substantia nigra of PD (n = 15) and DLB (n = 15) patients and age-matched non-demented controls (n = 15). Our results confirm elevated p-129 over total α-synuclein levels in the insoluble fraction of PD and DLB post-mortem brain tissue and we found significantly increased α-synuclein levels in the soluble fractions in PD and DLB. Furthermore, we identified an inverse correlation between reduced glucocerebrosidase enzyme activity and protein levels with increased glucosylsphingosine levels. In the substantia nigra, a brain region particularly vulnerable in Parkinson's disease, we found a significant correlation between glucocerebrosidase protein reduction and increased p129/total α-synuclein ratios. We assessed the direction and strength of the interrelation between all measured parameters by confirmatory path analysis. Interestingly, we found that glucocerebrosidase dysfunction impacts the PD-DLB status by increasing α-synuclein ratios in the substantia nigra, which was partly mediated by increasing glucosylsphingosine levels. In conclusion, we show that the introduced immuno-based assays enable the quantitative assessment of glucocerebrosidase and α-synuclein parameters in post-mortem brain. In the substantia nigra, reduced glucocerebrosidase levels contribute to the increase in α-synuclein levels and to PD-DLB disease manifestation partly by increasing its glycolipid substrate glucosylsphingosine. This interrelation between glucocerebrosidase, glucosylsphingosine and α-synuclein parameters supports the hypothesis that glucocerebrosidase acts as a modulator of PD-DLB