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

Neuronal silencing of dendoU expression severely impairs fly lifespan, development and motor performance. Since eyes and brain are the major components of Drosophila's head, where dendoU is predominantly expressed, we used the developing eye-specific GMR-GAL4, or the pan-neuronal elav-GAL4 drivers for dendoU tissue-specific silencing. GMR-GAL4 mediated dendoU RNAi in vivo did not produce any significant eye Unprocessed RNAs were run in lanes U. 1 bp-step ladders, derived from P1 or P2 hydrolysis, are fractionated in lanes L. Letters "a-g" point to cleavage products. Bottom: schemes of P1 and P2 sequences and cleavage site positions. Long arrows, short arrows and arrowheads point to preferential cleavage sites, sites cleaved less efficiently and minor sites, respectively (quantification in Supplementary Fig. 1b Table 2 ). Besides pupal lethality (17%) and eclosion defects (3%), a drastic reduction in median lifespan was observed (the median lifespan was 32.07 ± 0.43 days for control flies vs 2.05 ± 0.01 days for elav-G4 > dendoU RNAi flies) (Fig. 4b) . Moreover, adult flies displayed several abnormalities (Fig. 4c) . They appeared as immature animals bearing unexpanded wings, soft cuticle, unretracted ptilinum, dimpled dorsal thorax, and misoriented scutellar bristles. Remarkably, immature-looking flies also showed severe locomotor defects and marked uncoordination. Compared to control flies (Supplementary video 2), pan-neuronal interfered flies were quite unable to walk or stay upright, and usually became stuck in the culture medium immediately after eclosion. Impaired body posture and overall weakness were so severe that standard climbing assay could not be performed. Therefore, locomotor activity of these flies was assessed by evaluating voluntary locomotion on a horizontal surface (Supplementary video 3). As shown in the video, walking assays revealed substantial impairment of motor capacities. Flies were unable to move and promptly restore themselves to an upright position after being knocked down to the bottom of a dish by gentle agitation. The same immature phenotype and locomotor defects were observed in an independent RNAi line, upon exclusion of any off-target effect ( Supplementary Fig. 4a ,b and Supplementary video 4).