Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration

Laneve, Pietro; Piacentini, Lucia; Casale, Assunta Maria; Capauto, Davide; Gioia, Ubaldo; Cappucci, Ugo; Di Carlo, Valerio; Bozzoni, Irene; Di Micco, Patrizio; Morea, Veronica; Di Franco, Carmela Antonia; Caffarelli, Elisa

Expression profile and western blot analyses. mRNA and protein levels were determined by quantitative real-time PCR (qRT-PCR) and semi-quantitative PCR analysis (RT-PCR) or by Western blot assay, respectively. For qRT-PCR, RNA extracted from specific samples (developmental stages or tissues) by Qiazol reagent (Qiagen) was used to generate cDNA using SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen). The qRT-PCR was performed using QuantiFast SYBR Green PCR Kit (Qiagen) or miScript SYBR-Green PCR Kit (Qiagen) through a 7500 Fast Real-Time PCR (Applied Biosystem), using the oligonucletides listed below. The reaction mixtures were kept at 95 °C for 15 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Fluorescence output results were captured and analyzed using 7300 System SDS v1.4 Software (Applied Biosystems), and the threshold cycle (Ct) was used for assessing relative levels of target transcripts versus rp49 transcripts. For RT-PCR, amplicons were fractionated along a 2% agarose gel and revealed by ChemiDoc XRS + Molecular Imager (Bio-Rad). Gapdh mRNA was used as loading control. ImageJ was used for intensity band quantification. For protein level determination, Western blots were carried out using custom antibodies against CG2145 (PRIMM) (1:1000) and dTDP-43 18 (1:1000), or antibodies against Giotto (provided by G. Cestra) (1:5000). Walking, climbing and survival assays. The assays were performed according to earlier established protocol 18 . For the walking assay, we examined the behaviour of freely walking upright adult flies on a flat horizontal surface. Newly eclosed female and males were transferred in batches of 15 to fresh vials and aged for 3-4 days. The flies were transferred, without anaesthesia, to the centre of 200 mm Petri dish to allow for video recordings using a Nikon D5000 mounted on a WILD M3B (Wild Heerbrugg) stereomicroscope. For the climbing assay we used an apparatus consisting of two empty polystyrene vials vertically joined by tape facing each other. For the lower vial, a vertical distance of 8 cm above the bottom surface was measured and marked by drawing a circle around the entire circumference of the vial. For the climbing assay, a group of 10 male flies were transferred into the lower vial and allowed to acclimatize to the new setting for 1 minute. The climbing assay involved gently tapping the flies down to the bottom of the vial and measuring the number of flies per group that can climb above the 8 cm mark by 15 seconds after the tap, recorded as the percentage success rate. Nine trials were performed for each group and n ≥ 100 flies were assayed for each genotype. Experiments were performed during daylight to minimize potential effects of circadian oscillation. All average data are presented as mean ± SEM and compared with 2-tailed unpaired t-tests. Statistical tests were performed using Prism (GraphPad Software, Inc.). For survival assay, homozygous virgins bearing GAL4 were crossed to homozygous UAS-dendoU RNAi transgene and Scientific RepoRts | 7:41559 | DOI: 10.1038/srep41559 control Ore-R males. The progeny from these crosses were maintained on standard cornmeal-sucrose-yeast-agar diet at 29 °C. Every 2-3 days, flies were passed into new vials and dead flies were counted. The survival rate was calculated by the percentage of total flies surviving. Survival curves were analysed by the log-rank method using GraphPad Prism (n ≥ 500 flies for all genotypes except for D42-G4 >