Post-transcriptional silencing and functional characterization of the Drosophila melanogaster homolog of human Surf1

Abstract

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C-GAL4 or elav-GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C-GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav-GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C-GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav-GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Figure 1.

Expression analysis in Actin5C–GAL4 Surf1 KD larvae. (A) Northern blot analysis using as a probe the complete cDNA of the Surf1 gene. The 2-kb band is relative to the transcription of the hairpin construct while the intense smear, below the band at 0.9 kb (which corresponds to the Surf1 transcript), is indicative of degradation products resulting specifically from dsRNAi. This pattern of degradation is not seen when the Northern blots are hybridized with a probe against the heterologous GFP spacer which separates the two arms of the IR (data not shown). Signs of very slight aspecific mRNA degradation (probably artifactual) are also visible below the 2-kb band, as well as below the rp49 band in the samples collected from larvae, but not in those from adults. Positive controls consisted in mRNA extracted from larvae of the w¹¹¹⁸ strain, which was used to generate the UAS–Surf1 inverted repeat transgenic lines, for which two different quantities of mRNA (25 and 50 μg) were loaded on the gels. The experiments with the KD individuals were done using 50 μg of mRNA. In each case a housekeeping gene, i.e., rp49 (which encodes for Drosophila ribosomal protein 49), served as an external reference. (B) Real-time PCR estimate of the relative percentage of Surf1 mRNA in KD individuals from each of the three lines analyzed compared to those of the respective parental lines bearing only the UAS–Surf1-inverted repeat (controls). In each case controls are assigned an arbitrary value of 100%. Histograms represent the mean of two independent experiments. Values obtained for each independent experiment were: 32 and 43 for Act–GAL4 KD^79.1, 12.5 and 36 for Act–GAL4 KD^79.10, 7.5 and 12.2 for Act–GAL4 KD^23.4. (C) Western blot analysis on KD and control (see B) individuals. The 70-kDa band corresponds to an unknown protein, which was used as a reference signal for the quantization of the SURF1 signal. Controls consisted of mitochondrial protein extracts from larvae, pupae or adults of the w¹¹¹⁸ strain (see above).

Figure 2.

In situ expression analysis of Surf1 mRNA in whole-mount preparations of wild-type embryos. (A) Stage 9, negative control. (B) Stage 4–5, blastoderm. (C) Stage 9–10. (D) Stage 14–15. In all developmental stages analyzed expression is fairly strong and ubiquitous, with a tendency to become weaker toward the end of embryonic development (see D).

Figure 3.

Relative percentage of egg-to-adult viability, calculated at each of four developmental stages, i.e., eggs, larvae, pupae and adults, in controls (black) and Actin5C–GAL4-driven Surf1 knockdown flies (white). For each line, the control consisted of individuals from the line bearing the non-activated UAS–Surf1 inverted repeat. CS, Canton-S, a reference wild-type strain.

Figure 4.

Most of the Actin5C–GAL4 Surf1 KD individuals die as larvae, which show an impaired development: 7 day-old KD larvae (B) appear drastically smaller than controls (A) and have undersized optic lobes (C is a control brain, D is a brain from Actin5C–GAL4-driven Surf1 KD). KD larvae from lines 23.4 and 79.10 have all the distinctive characters of the third instar (i.e., the cuticular structures: mouth hooks (E) and anterior spiracles (F); see red arrows). On the other hand, in Actin5C–GAL4 Surf1 KD^79.1 larvae, cuticular structures are typical of the second instar (G and H); see red arrows. Some KD larvae reach the pupal stage and do not progress any further in development. Dissection of the dead pupae shows that individuals die at early imago stages, when adult cuticular structures have only just everted. I and J are control pupae, respectively, with and without puparium; K and L are Actin5C–GAL4 Surf1 KD pupae.

Figure 5.

(A) The speed of locomotion (cm/sec) measured in Actin5C–-GAL4 Surf1 KD larvae is significantly different from that of the respective controls for both lines analyzed (23.4 vs. control, P < 0.0001, n = 23 and 21, respectively; 79.10 vs. control, P = 0.0366, n = 20 and 10, respectively). (B) Response in the photobehavioral assay of Surf1 KD and control larvae tested using the checker test (see materials and methods). Briefly, the photobehavioral test consists of the evaluation of the larval light-avoidance reaction. In the checker test paradigm this is evaluated by the ratio, expressed in the form of a normalized relative index (RI), of the time spent by a larva on the black (opaque to light) squares with respect to the white ones (transparent to light) of a checkerboard illuminated from beneath. KD individuals from both lines showed a significant decrease in their ability to avoid the light stimulus [Act–GAL4 KD^23.4 vs. control (UAS–IR^23.4); P = 0.032] and [Act–GAL4 KD^79.10 vs. control (UAS–IR^79.10); P = 0.02]. (C and D) An example of phalloidin-rhodamine staining of whole-mount larval body-wall preparations shows no major anomalies in muscle structure and arrangement in Actin5C–GAL4 Surf1 KD^79.1 individuals (D), although a clear reduction in size is evident with respect to controls (C). (E and F) Cross sectional ultrastructure of glutamatergic synapses from third segment muscles 6 or 7 of stage three larvae. (E) Electron micrograph of a Type 1 bouton in a control (UAS–IR^23.4) larva, showing the well-developed subsynaptic reticulum (SSR); see arrows. (F) Representative micrograph of a Type 1 bouton in an Actin5C–GAL4 Surf1 KD^23.4 larva, showing a reduction in the complexity of the SSR, which is more typical of a mid first to second stage wild type larva. Magnification (E and F), ×2842.

Figure 6.

Electron micrograph of larval body-wall muscle fibers showing mitochondria (arrows) in control larvae bearing only the UAS–Surf1 inverted repeat and in Actin5C–GAL4 KD individuals. Cross section of control (A) and Actin5C–GAL4 KD (B) larva. Longitudinal section of control (C) and Actin5C–GAL4 KD (D) larva. Magnification for all figures: ×10,000. Mitochondria in controls tend to be rather small, elongated, and homogeneously stained, forming clusters within the intermyofibrillar spaces, whereas mitochondria in KD individuals are much larger and round with an apparently disorganized internal structure (i.e., matrix and cristae). Furthermore, the latter mitochondria are not typically found in clusters within the muscle fiber.

Figure 7.

Graphs showing the number of adult flies surviving after up to 115 days. (A) w¹¹¹⁸ (the background used for transgenesis) and elav–GAL4 x w¹¹¹⁸. (B) 23.4 Surf1 IR and elav–GAL4 KD^23.4. (C) 79.10 Surf1 IR and elav–GAL4 KD^79.10. (D) 79.1 Surf1 IR and elav–GAL4 KD^79.1. For each graph the statistical comparison of the pairs of curves (i.e., KD vs. parental IR line) was done using the Wilcoxon test. (A) P = NS; (B) P = 0.008; (C) P = 0.02; (D) P = NS.

Figure 8.

(A) COX activity in adult control flies bearing only the UAS–Surf1 inverted repeat. (B) COX activity in elav–GAL4 Surf1 KD adult flies from line 23.4. (C) Succinate dehydrogenase (SDH) activity in adult control flies bearing only the UAS–Surf1 inverted repeat. (D) SDH activity in elav–GAL4 Surf1 KD adult flies from line 23.4. Nuclear-encoded SDH is localized to the internal mitochondrial membrane. Thus, comparison of COX levels to those of SDH provide an indication of the contribution of COX activity relative to SDH, an independent indicator of mitochondrial mass. In each image analyzed, the ROI were limited to the optic neuropils (i.e., lamina, medulla, lobula, and lobula plate). ROIs were identified in the digitized images (i.e., the single neuropils), and pixel counts and gray level distributions were performed within these ROIs. The mean gray level/pixel for each ROI was then used as an estimate of the level of enzyme activity being determined (i.e., high mean gray level/pixel = high enzyme activity). Sections obtained from heads of elav–GAL4 KD^23.4 flies showed a significant (Student's t-test, P < 0.05) reduction in COX activity [KD^23.4 = 177.98 ± 22.03 vs. controls (UAS–IR^23.4) = 189.23 ± 15.43] and a highly significant (Student's t-test, P < 0.001) increase in SDH activity [KD^23.4 = 171.514 ± 25.34 vs. controls (UAS–IR^23.4) = 140.60 ± 31.27]. In particular SDH activity in KD individuals is greater than that observed in controls, suggesting an increase in mitochondrial mass in the former. La, lamina; Me, medulla; Lo, lobula; Lp, lobula plate.

Figure 9.

Hematoxilin-eosin stains of paraffin-embedded frontal cephalic sections of adult flies. (A) Control flies bearing only the UAS-Surf1 inverted repeat. (B) elav–GAL4 Surf1 KD adult flies from line 23.4. Me, medulla; Lo, lobula; Lp, lobula plate; Al, antennal lobe; vlP, ventrolateral protocerebrum; mP, medial protocerebrum. Arrows indicate sectioning artifacts.

Figure 10.

(A) Optomotor behavior in elav–GAL4 Surf1 KD and control (UAS–IR) adults. KD flies (males and females) from lines 23.4 and 79.1 and only KD males from line 79.10 turned at random, giving mean values of correct turns not significantly different from 50%. On the other hand, controls (UAS–IR^23.4, UAS–IR^79.1 and UAS–IR^79.10) turned in the direction of the moving environment 70–80% of the time. For each genotype 20 individuals (10 males and 10 females) were tested. Each fly was given 10 trials, and each time the direction of rotation of the stripes was changed. (B, top) An example of a wild-type ERG response showing the major features, the ON and OFF transients with the sustained response between the two transients. (B, bottom) Example of an ERG response from an elav–GAL4 KD individual from line 79.1; in this case the ON and OFF transients are completely missing. (C, D, and E) results of ERGs recorded from elav–GAL4 Surf1 KD individuals from lines 23.4 and 79.1 and controls (as in A). Graphs represent mean amplitudes (mV ± SD) of ON (C) and OFF (D) transients (due to synaptic activation of second order neurons of the visual pathway) and of the sustained response (E) (due to photoreceptor light-induced depolarization). Mean amplitude of ON and OFF transients for both lines of KD vs. control (as above) individuals were significantly different (P < 0.05). However, only the sustained response of KD individuals from line 79.1 was significantly different to the relative control (as above). The graphs showing the ERG data are relative to 16–29 individuals for line 23.4 and 11–14 individuals in the case of line 79.1.