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^RNAi</i>) and elav<i>>mort^GD</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 µm. (<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 “Green-Fire-Blue” allows the separation of autophagosomes from the diffuse ATG8-mRFP signal. Scale bar: 10 µm. (<b>C</b>) Confocal images of synaptic boutons at NMJ 4 in control (elav<i>>white^RNAi</i>) and elav<i>>mort^GD</i> larvae. Neuronal membranes and autophagosomes are shown in green and magenta, respectively. Scale bar: 5 µm. (<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 Student’s t-test.</p

Knockdown of <i>Hsc70-5/mortalin</i> Induces Loss of Synaptic Mitochondria in a <i>Drosophila</i> Parkinson’s 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 Parkinson’s 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

<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 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. <i>mortalin</i> expression was repressed using two UAS-RNAi stocks named <i>mort^GD47745</i> (<i>mort^GD</i>) and <i>mort^KK106236</i> (<i>mort^KK</i>). In <i>mort^GD</i> (purple arrow) and <i>mort^KK</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, 29°C) of <i>mort^GD</i> and <i>mort^KK</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 29°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^RNAi</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^KK</i> but not <i>elav>mort^GD</i> larvae. Statistical significance was determined using a Kruskal-Wallis H-test followed by Dunn’s 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^GD</i> larvae at rest or during locomotion. Scale bar: ∼0.25 mm (<b>C–G</b>) 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 <i>mort^GD</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 µm. Statistical significance was determined using an unpaired, two-tailed Student’s t-test.</p

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^RNAi</i>) and elav><i>mort^GD</i> larvae. Neuronal membranes (HRP), autophagosomes, and mito-GFP are shown. In elav<i>>mort^GD</i> larvae, mitochondria frequently co-localized with autophagosomes. Scale bar: 10 µm, Enlargement: 2 µm (<b>B</b>) The number of mitochondria and autophagosomes per NMJ is shown. Most autophagosomes in elav<i>>mort^GD</i> larvae co-localized with mitochondria, either by being directly adjacent or overlapping. (<b>C</b>) In human fibroblasts (n = 56 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 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.</p

Establishment of a <i>Drosophila</i> SPG 10 model.

<p>(A) Khc protein alignment across species (<i>Danio rerio</i> (Dr), <i>Tribolium castaneum</i> (Tc), <i>Xenopus tropicalis</i> (Xt), <i>Drosophila melanogaster</i> (Dm), <i>Homo sapiens</i> (Hs), <i>Rattus norvegicus</i> (Rn)) shows unique conservation of the amino acids spanning from the β-sheet 7 (β7) to the α-helix 4 (α4). Pathological mutations (red dots) are highly enriched in the latter half of loop 11 (L11) that links β7 to α4. The human mutation at position 256 (red star) was selected for further analysis. Accession numbers for the proteins used in the alignment are: Dr: GenBank_CAQ15489.1; Tc: EFA10675.1; Xt: NCBI_NP_001096215.1; Dm: GenBank_AAF58029.1, Hs: NCBI_NP_004975.2; Rn: EDM 16486.1 (B) 3D protein structure of rat Khc (3KIN) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066-Kozielski1" target="_blank">[8]</a>. The microtubule binding site is shown in yellow and green. L11 (brown cylinder), which was not resolved in the crystal structure, was added between β7 (yellow) and α4 (pink). (C) Analysis of larval locomotion. Larvae overexpressing D42>Khc^N262S (25°C) are almost completely (excluding the head) paralyzed (red arrow), whereas D42>Khc^wt+N262S larvae are still crawling, but display a tail-flipping phenotype (green arrowhead). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s007" target="_blank">Videos S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s008" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s009" target="_blank">S3</a>) (D) Kaplan-Meier survival curve recorded at 18°C (Genotypes: black: D42>w¹¹¹⁸; Green: D42>Khc^wt; Blue: D42>Khc^N262S; Red: D42>Khc^wt+N262S) (E) Summary of the data derived from the survival analysis shown in (D). Statistical significance of data was determined by a series of Mantel-Cox tests. *** p<0.001.</p

Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a <em>Drosophila</em> HSP Model

<div><p>Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a <em>Drosophila</em> model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of <em>Drosophila</em> Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in <em>Drosophila</em>. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.</p> </div

Characterization of adult <i>Drosophila</i> SPG 10 model flies.

<p>(A) Analysis of adult wing posture. While control flies have a normal wing posture (green arrowheads), Khc^N262S expressing flies frequently display abnormal wing postures such as holding their wings up (red arrow), suggesting either the degeneration or functional impairment of the indirect flight muscles. 2-day old male flies raised at 18°C are shown. All flies contained a copy of the motoneuron specific driver D42-Gal4. (B) Analysis of thorax muscle integrity provided no evidence for the degeneration of the indirect flight muscles. Scale bar: 100 µm. (C) Western blot of whole fly head extracts after adult-onset pan-neuronal (elav^C155-Gal4/tub-gal80ts) expression of Khc^N262S and Khc^wt at 29°C for 13 days. Levels of endogenous Khc from 10 heads of control flies (w¹¹¹⁸) are comparable to Khc levels in 2.5 heads from flies overexpressing Khc (Khc^N262S and Khc^wt). The number of heads loaded per lane is indicated. β-Tubulin was used as loading control. (D) Climbing assay of adult flies 16 days after adult-onset pan-neuronal expression of Khc (white bars). Although Khc^wt expression caused no adverse effects on climbing scores compared with control (grey bars), Khc^N262S expression significantly lowered the climbing score. Statistical significance was tested using an unpaired, two-tailed student's t-test (*** p<0.001). The standard error of the mean (s.e.m) is shown as a box, standard deviation (s.d.) as a black line.</p

Expression of Khc^N262S causes length-dependent synaptic defects at the neuromuscular junction.

<p>Confocal images and quantification of immunofluorescent staining of synaptic marker proteins. NMJs 6/7, segment A5 of mid-third-instar <i>Drosophila</i> larva, were selected for analysis. D42>w¹¹¹⁸ and D42>khc^wt larvae were used as controls for the mutant phenotype D42>khc^N262S. All three selected synaptic marker proteins were reduced in abundance at the NMJs of khc^N262S-expressing larvae. Larvae were raised at 29°C. (A,B) The active zone protein Brp, as well as the synaptic vesicle proteins (C,D) DV-Glut and (E,F) CSP, were selected as synaptic marker proteins. (A,C,E) Confocal images revealed that DV-Glut and CSP abundance is increased in dystrophic boutons (C,E, arrows) and reduced elsewhere (C,E, arrowheads) at NMJs of D42>khc^N262S larvae. Scale bar: 5 µm; inset 2.5 µm. (B,D,F) For quantification, n = 8–10 NMJs were analyzed per genotype. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. * p<0.05; ** p<0.01.</p

Neuromuscular function.

<p>Evoked junctional potentials (EJPs), the half width of EJPs and mini excitatory junctional potentials (mEJPs) were determined in D42>w¹¹¹⁸, D42>khc^N262S and D42>khc^wt+N262S larvae. (A) Averaged traces of EJPs. Stimulation artifacts were removed. (B) Quantification of averaged EJP size. (C) Half width of EJPs. (D) Averaged amplitudes of mEJPs from control and mutant larvae. (E) Quantification of averaged mEJP size. (F) Quantification of the number of vesicles released per action potential. Statistics n = 8–9 larvae per genotype for B,C,E,F. One-way ANOVA followed by Tukey-Kramer post-test ** p<0.01; *** p<0.001. The standard error of the mean (s.e.m) is shown as a box, the standard deviation (s.d.) as a black line.</p