5 research outputs found
Pan-neuronal knockdown of <i>mortalin</i> induced autophagy at the larval NMJ.
<p>(<b>A</b>) <i>Drosophila</i> VNCs of control (elav<i>>white<sup>RNAi</sup></i>) and elav<i>>mort<sup>GD</sup></i> larvae labeled with the autophagosomal ATG8-mRFP marker. No obvious change in the ATG8-mRFP signal was detected upon <i>mortalin</i> knockdown. Gamma values were adjusted to 0.75 Scale bar: 50 Āµm. (<b>B</b>) Autophagosomes were detected as the strong accumulation of ATG8-mRFP signal at the <i>Drosophila</i> NMJ. The false color look-up table āGreen-Fire-Blueā allows the separation of autophagosomes from the diffuse ATG8-mRFP signal. Scale bar: 10 Āµm. (<b>C</b>) Confocal images of synaptic boutons at NMJ 4 in control (elav<i>>white<sup>RNAi</sup></i>) and elav<i>>mort<sup>GD</sup></i> larvae. Neuronal membranes and autophagosomes are shown in green and magenta, respectively. Scale bar: 5 Āµm. (<b>D, E</b>) Statistical analysis revealed increases in ATG8-mRFP puncta abundance (<b>D</b>) and size (<b>E</b>). Statistical significance was determined by using an unpaired, two-tailed Studentās t-test.</p
Knockdown of <i>Hsc70-5/mortalin</i> Induces Loss of Synaptic Mitochondria in a <i>Drosophila</i> Parkinsonās Disease Model
<div><p>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 Parkinsonās disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, <i>Drosophila melanogaster</i> has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-<i>Hsc70-5/mortalin</i>-function <i>Drosophila</i> model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing <i>Drosophila</i> 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 <i>mortalin</i> 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 <i>mortalin</i>-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. <i>Ex vivo</i> 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.</p></div
Loss of <i>mortalin</i> function induces mitophagy.
<p>(<b>A</b>) Confocal images of NMJ 4 at Segment A5 of the mid third instar larvae in control (elav><i>white<sup>RNAi</sup></i>) and elav><i>mort<sup>GD</sup></i> larvae. Neuronal membranes (HRP), autophagosomes, and mito-GFP are shown. In elav<i>>mort<sup>GD</sup></i> larvae, mitochondria frequently co-localized with autophagosomes. Scale bar: 10 Āµm, Enlargement: 2 Āµm (<b>B</b>) The number of mitochondria and autophagosomes per NMJ is shown. Most autophagosomes in elav<i>>mort<sup>GD</sup></i> larvae co-localized with mitochondria, either by being directly adjacent or overlapping. (<b>C</b>) In human fibroblasts (nā=ā56 cells) the mitochondrial-lysosomal colocalization was higher in cells from a carrier of the loss of <i>mortalin</i> 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 Āµm and 2 Āµm. 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 Studentās t-test.</p
<i>Hsc70-5</i> (<i>CG8542, mortalin</i>) is a <i>Drosophila</i> homolog of the PD-associated gene <i>mortalin</i>.
<p>(<b>A</b>) The genomic organization of <i>Hsc70-5</i> (<i>CG8542, mortalin</i>) 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 5ā²-UTR and 3ā²-UTR are shown as a gray box and a gray triangle, respectively. The exact sequence location (2R:10,140,103ā¦10,143,697 [ā]) is given at the top of the panel. <i>mortalin</i> expression was repressed using two UAS-RNAi stocks named <i>mort<sup>GD47745</sup></i> (<i>mort<sup>GD</sup></i>) and <i>mort<sup>KK106236</sup></i> (<i>mort<sup>KK</sup></i>). In <i>mort<sup>GD</sup></i> (purple arrow) and <i>mort<sup>KK</sup></i> (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 <i>mortalin</i> were expressed. These double-stranded RNAs are processed into short siRNAs that are predicted to induce <i>mortalin</i> mRNA degradation. (<b>B</b>) <i>Drosophila</i> Mortalin (black box) has a high sequence similarity with human Mortalin. The 686-amino acid-long <i>Drosophila</i> 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 <i>Drosophila</i> mortalin is the highest in the central domain of the protein. (<b>C</b>) The ubiquitous and pan-neuronal knockdown of <i>mortalin</i> resulted in larval and pupal lethality, while <i>mortalin</i> knockdown in muscle did not impair viability. (<b>D</b>) 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, 29Ā°C) of <i>mort<sup>GD</sup></i> and <i>mort<sup>KK</sup></i> (<b>E</b>) Eye-specific knockdown of <i>mortalin</i> 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 29Ā°C. Scale bar: 0.1 mm (<b>F</b>) <i>Mortalin</i> deficiency in DA neurons is lethal, whereas GMR- and ey- driven expression of <i>mortalin<sup>RNAi</sup></i> does not affect viability. Knockdown of <i>mortalin</i> 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. <i>mortalin</i> knockdown led to lethality with most GAL4 drivers that induce expression in motoneurons (OK6-, OK371-, D42-GAL4).</p
Quantification of synaptic terminals in <i>mortalin</i> knockdown larvae.
<p>(<b>A</b>) 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 <i>mortalin</i> silencing impaired locomotor function of <i>elav>mort<sup>KK</sup></i> but not <i>elav>mort<sup>GD</sup></i> larvae. Statistical significance was determined using a Kruskal-Wallis H-test followed by Dunnās test for comparisons between multiple groups. (<b>B</b>) Analysis of larval crawling did not reveal any body-posture defect of 4-day old mid-L3 stage <i>elav>mort<sup>GD</sup></i> larvae at rest or during locomotion. Scale bar: ā¼0.25 mm (<b>CāG</b>) Confocal images of NMJ 4 at Segment A5 of the mid third instar larvae raised at 29Ā°C. Visualization of neuronal membranes marked with HRP-Cy3 allowed assessment of NMJ morphology. Pan-neuronal expression of <i>mort<sup>GD</sup></i> did not affect (<b>D</b>) muscle length, (<b>E</b>) NMJ size, or the number (<b>F</b>) or size (<b>G</b>) of synaptic boutons. Scale bar: 5 Āµm. Statistical significance was determined using an unpaired, two-tailed Studentās t-test.</p