Knockdown of Hsc70-5/mortalin induces loss of synaptic mitochondria in a Drosophila Parkinson's disease model

Abstract

Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson's disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.

Figure 1. Hsc70-5 (CG8542, mortalin) is a Drosophila homolog of the PD-associated gene mortalin.

(A) The genomic organization of Hsc70-5 (CG8542, mortalin) located on the second chromosome at cytological position 50E6. Genes and transcripts are displayed in blue and gray/yellow, respectively. Coding exons are depicted as yellow boxes, the 5′-UTR and 3′-UTR are shown as a gray box and a gray triangle, respectively. The exact sequence location (2R:10,140,103…10,143,697 [−]) is given at the top of the panel. mortalin expression was repressed using two UAS-RNAi stocks named mort^GD47745 (mort^GD) and mort^KK106236 (mort^KK). In mort^GD (purple arrow) and mort^KK (cyan arrow), 303-bp and 415-bp-long hairpin RNAs directed against gene fragments located to two partially overlapping domains in the fifth exon of mortalin were expressed. These double-stranded RNAs are processed into short siRNAs that are predicted to induce mortalin mRNA degradation. (B) Drosophila Mortalin (black box) has a high sequence similarity with human Mortalin. The 686-amino acid-long Drosophila Mortalin protein shares overall 73% identity and 84% similarity with the 679-amino acid-long human Mortalin. The percent homology, color coded in the bottom panel, between human and Drosophila mortalin is the highest in the central domain of the protein. (C) The ubiquitous and pan-neuronal knockdown of mortalin resulted in larval and pupal lethality, while mortalin knockdown in muscle did not impair viability. (D) The protein level of Mortalin in the ventral nerve cord (VNC) of mid third instar larvae was measured by western blot upon pan-neuronal expression (elav-GAL4, 29°C) of mort^GD and mort^KK (E) Eye-specific knockdown of mortalin did not cause visible defects in the external adult eye of the young and ageing flies. All the flies carrying the induced RNAi constructs were raised at 29°C. Scale bar: 0.1 mm (F) Mortalin deficiency in DA neurons is lethal, whereas GMR- and ey- driven expression of mortalin^RNAi does not affect viability. Knockdown of mortalin in DA neurons using Ddc- or TH-GAL4 resulted in lethality during larval or pupal stages; no effect was seen following knockdown in sensory neurons. mortalin knockdown led to lethality with most GAL4 drivers that induce expression in motoneurons (OK6-, OK371-, D42-GAL4).

Figure 2. Analysis of the effects of housekeeping gene knockdown in Drosophila eye.

(A) The eye-specific knockdown of Drosophila housekeeping genes resulted in diverse phenotypes. Examination of eyes revealed the effect of GMR-GAL4 driven RNAi silencing at 29°C. (B) GMR>mort^KK did not cause degeneration in the external eyes of adult flies compared to the GMR-GAL4 (control), which displayed minor basal toxicity compared to uninduced flies (mort^KK). The eye-specific inactivation of some Drosophila housekeeping genes induced strong degeneration. The arrowheads point to the black lesions indicative of necrosis. Scale bar: 0.1 mm.

Figure 3. Pan-neuronal knockdown of mortalin caused behavioral defects and reduced adult Drosophila lifespan.

(A) Kaplan-Meier survival curve recorded at 18°C. Lifespan reduction was detected upon pan-neuronal (elav-GAL4) mortalin knockdown. Female flies were examined. Statistical significance of the data was determined by a series of Mantel-Cox tests. (B) Walking tests showed that pan-neuronal mortalin silencing resulted in reduced locomotor function. All the flies were raised at 18°C. Statistical significance was determined using an unpaired, two-tailed Student’s t-test. (C) Characteristic wing posture phenotype caused by the weak pan-neuronal expression of mortalin^RNAi. The top image displays the normal wings of control (elav>white^RNAi) flies; the bottom picture shows the abnormal wing posture of elav>mort^GD-expressing flies. All the flies were raised at 18°C. Scale bar: 0.25 mm (D) Climbing tests were used to assess locomotor behavior. Statistical significance was determined by using a Kruskal-Wallis H-test followed by a Dunn’s test for comparisons among multiple groups. (E) The characteristic wing posture phenotype caused by the pan-neuronal expression of mortalin RNAi. Pan-neuronal mortalin silencing in mort^KK resulted in an increased wing phenotype percentage. (F) ATP level was measured in the heads of 4-day old female flies. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn’s test for comparisons between multiple groups.

Figure 4. Quantification of synaptic terminals in mortalin knockdown larvae.

(A) Larvae locomotor behavior and body posture control were assessed with the righting assay in 4-day old mid-L3 stage larvae. The average righting time is determined for larvae placed upside down on agar plate. Pan-neuronal mortalin silencing impaired locomotor function of elav>mort^KK but not elav>mort^GD larvae. Statistical significance was determined using a Kruskal-Wallis H-test followed by Dunn’s test for comparisons between multiple groups. (B) Analysis of larval crawling did not reveal any body-posture defect of 4-day old mid-L3 stage elav>mort^GD larvae at rest or during locomotion. Scale bar: ∼0.25 mm (C–G) Confocal images of NMJ 4 at Segment A5 of the mid third instar larvae raised at 29°C. Visualization of neuronal membranes marked with HRP-Cy3 allowed assessment of NMJ morphology. Pan-neuronal expression of mort^GD did not affect (D) muscle length, (E) NMJ size, or the number (F) or size (G) of synaptic boutons. Scale bar: 5 µm. Statistical significance was determined using an unpaired, two-tailed Student’s t-test.

Figure 5. Quantification of mitochondria in Drosophila larvae upon silencing of mortalin expression.

(A) Confocal images of synaptic boutons in control (elav>white^RNAi) and elav>mort^GD larvae. The membrane marker HRP-Cy3 is shown in green, and native fluorescence of mito-GFP is shown in magenta. Scale bar: 5 µm. mortalin silencing significantly reduced (B) the number of mitochondria per NMJ, (C) the area fraction of the NMJ positive for mitochondria. Furthermore, the average size (D) of mitochondria was reduced, while the fraction of circular mitochondria (E) was increased. Statistical significance was determined using an unpaired, two-tailed Student’s t-test.

Figure 6. Pan-neuronal knockdown of mortalin induced autophagy at the larval NMJ.

(A) Drosophila VNCs of control (elav>white^RNAi) and elav>mort^GD larvae labeled with the autophagosomal ATG8-mRFP marker. No obvious change in the ATG8-mRFP signal was detected upon mortalin knockdown. Gamma values were adjusted to 0.75 Scale bar: 50 µm. (B) Autophagosomes were detected as the strong accumulation of ATG8-mRFP signal at the Drosophila NMJ. The false color look-up table “Green-Fire-Blue” allows the separation of autophagosomes from the diffuse ATG8-mRFP signal. Scale bar: 10 µm. (C) Confocal images of synaptic boutons at NMJ 4 in control (elav>white^RNAi) and elav>mort^GD larvae. Neuronal membranes and autophagosomes are shown in green and magenta, respectively. Scale bar: 5 µm. (D, E) Statistical analysis revealed increases in ATG8-mRFP puncta abundance (D) and size (E). Statistical significance was determined by using an unpaired, two-tailed Student’s t-test.

Figure 7. Loss of mortalin function induces mitophagy.

(A) Confocal images of NMJ 4 at Segment A5 of the mid third instar larvae in control (elav>white^RNAi) and elav>mort^GD larvae. Neuronal membranes (HRP), autophagosomes, and mito-GFP are shown. In elav>mort^GD larvae, mitochondria frequently co-localized with autophagosomes. Scale bar: 10 µm, Enlargement: 2 µm (B) The number of mitochondria and autophagosomes per NMJ is shown. Most autophagosomes in elav>mort^GD larvae co-localized with mitochondria, either by being directly adjacent or overlapping. (C) In human fibroblasts (n = 56 cells) the mitochondrial-lysosomal colocalization was higher in cells from a carrier of the loss of mortalin function variant compared with cells from a healthy sibling control. Colocalization is indicated by a yellow signal due to overlapping Lysotracker red and Mitotracker green staining. Scale bar: 10 µm and 2 µm. Statistical analysis revealed a higher number of mitochondria colocalized with lysosomes in the mutant compared with control cells. Statistical significance was determined using an unpaired, two-tailed Student’s t-test.