Impairment of Drosophila orthologs of the human orphan protein C19orf12 induces bang sensitivity and neurodegeneration

Abstract

Mutations in the orphan gene C19orf12 were identified as a genetic cause in a subgroup of patients with NBIA, a neurodegenerative disorder characterized by deposits of iron in the basal ganglia. C19orf12 was shown to be localized in mitochondria, however, nothing is known about its activity and no functional link exists to the clinical phenotype of the patients. This situation led us to investigate the effects of C19orf12 down-regulation in the model organism Drosophila melanogaster. Two genes are present in D. melanogaster, which are orthologs of C19orf12, CG3740 and CG11671. Here we provide evidence that transgenic flies with impaired C19orf12 homologs reflect the neurodegenerative phenotype and represent a valid tool to further analyze the pathomechanism in C19orf12-associated NBIA.

Figure 1. Alignment of the fly C19orf12 orthologs (CG3740, CG11671) with human C19orf12 protein.

Proteins have been aligned using Clustal 2.1 multiple alignment tool. Stars (*) indicate identities and dots indicate a higher (:) and a lower (.) degree of similarity. The two D. melanogaster proteins are 63% and 55% similar to C19orf12 and share 72% similarity with each other. The transmembrane domains predicted by PolyPhobius are marked in yellow.

Figure 2. Expression of CG3740 and CG11671 in head, thorax and abdomen of D. melanogaster.

The analysis has been performed on total RNA extracted from adult w¹¹¹⁸ flies using the same number of males and females (n = 4). The Ribosomal Protein 49 has been used as endogenous control to normalize CG3740 and CG11671 expression level. The level of CG11671 in the abdomen has been set to 1 and the expression of CG3740 and CG11671 in the other tissues expressed relatively.

Figure 3. Expression of CG3740 and CG11671 in heads from 10 days-old flies.

The Ribosomal Protein 49 has been used as endogenous control to normalize CG3740 and CG11671 expression level. (A) CG3740 and CG11671 have been measured in single RNAi flies (CG3740 RNAi and CG11671 RNAi) and in double RNAi flies (CG3740; CG11671 RNAi) using the same number of males and females (n = 4). The level of CG3740 and CG11671 in control flies has been set to 1 and the expression of both genes in down-regulated flies expressed relatively. (B) CG3740 and CG11671 have been measured in single heterozygous deletion flies (Df(1)BSC589/+ and Df(3L)BSC579/+) and in double heterozygous deletion flies (Df(1)BSC589/+; Df(3L)BSC579/+) using female flies (n = 4). The level of CG3740 and CG11671 in control flies has been set to 1 and the expression of both genes in heterozygous deletion flies expressed relatively. Data refer to the average of three independent experiments ± SD.

Figure 4. Kaplan-Maier curve of control (w¹¹¹⁸) and double RNAi male flies (n = 100).

The median lifespan is 33 days for control flies vs. 25 days for down-regulated flies. The difference was statistically evaluated by Log Rank test (p = 0.02).

Figure 5. Climbing activity in young (1 day-old) and aged (1 month-old) control (w¹¹¹⁸ and act-GAL4/+), double RNAi and double heterozygous flies.

The average is given for 50 animals in each group.

Figure 6. Prussian blue staining for detection of iron deposits.

The staining was performed on paraffin-embedded sections of 28 days-old flies. No iron deposits were detected in the medulla or retina of either controls or double RNAi flies. Clear iron inclusions were present in paraffin-embedded sections of mouse spleen (positive control).

Figure 7. Detection of vacuoles in ultrathin Epon plastic sections.

A) Horizontal head sections from 28 days-old control (w¹¹¹⁸) and down-regulated (CG3740; CG11671 RNAi) flies. B) Number and size of vacuoles have been quantified with Image J running the applications “Find Edges” and “Analyze Particles” on thresholded 8-bit pictures. Particle size was imposed bigger than 20 pixels² and with a circularity factor between 0,5 and 1. Detected vacuoles were displayed using “Overlay Mask”. Data were manually validated to exclude artifacts. Brains analyzed for each fly strain: n = 3. Scale bar: 100 µm.

Figure 8. Bang test in young (1 day-old) and aged (1 month-old) control (w¹¹¹⁸ and act-GAL4/+) and double RNAi males.

A) Flies have been vortexed twice (Bang I and II) with 10 minutes in between and the time needed to upright recorded. A) Representative trajectories are reported for aged control (green), double RNAi (red) and for double heterozygous (blue) flies.