Article Figures & Data
Figures
- Figure 1. Expression of pdzd8-RNAi reduces mitochondria-ER contacts.
(A) Domain organisation of Drosophila pdzd8 (CG10362) compared with mouse Pdzd8 showing percentage identities of conserved domains based on Clustal Omega alignments. Overall percentage identity of the amino acid sequences is 21%. SMP (synaptotagmin-like mitochondrial lipid-binding proteins) 33% identical, PDZ (PSD95/DLG/ZO-1) 36% identical, C1 (C1 protein kinase C conserved region 1 also known as Zn finger phorbol-ester/DAG-type signature) 47% identical; TM: predicted transmembrane domain, CC: coil–coil domain. (B) Electron microscopy images of cell bodies of the posterior protocerebrum of 2-d-old adult brains showing representative images of ER, mitochondria, and MERCs in soma from nSyb>LacZ-RNAi and nSyb>pdzd8-RNAi flies. Scale bar 500 nm. Mitochondria without identifiable ER contacts marked with magenta *, mitochondria forming ER contact marked with yellow * with yellow arrow indicating contact location, organelles that did not contain clear cristae are marked with a cyan * and were excluded from the analysis. (C) Percentage of mitochondria in contact with the ER from controls and pan-neuronal nSyb>pdzd8-RNAi flies quantified from EM images of 2-d-old adult brains. n = 3 brains per genotype, numbers on bars indicate number of mitochondria analysed. (D) SPLICS puncta indicating MERCs in axon bundles of larval neurons from controls and nSyb>pdzd8-RNAi flies. Quantified puncta highlighted with V. Scale bar 5 μm. (D, E) Quantification of SPLICS puncta in (D). n = 11 animals per genotype, P = 0.0198, unpaired t test with Welch’s Correction. (F) Representative structured illumination microscopy images of ER (green, ER-Tomato) and mitochondria (purple, mitoGFP) in larval epidermal cells from controls and da>pdzd8-RNAi flies. Scale bar = 5 μm. (F, G) Binarized images of ER and mitochondria shown in (F). (H) Quantification of colocalization of ER and mitochondria using Mander’s Correlations in 12 control cells and 14da > pdzd8-RNAi cells (1 field of view per cell) compared using an unpaired t test with Welch’s Correction. P = 0.012.
- Figure S1. Expression and tissue specificity of pdzd8.
(A) Tissue-specific expression of pdzd8 in 7-d-old adult males from Leader et al (2018). FPKM, fragments per kilobase of exon model per million reads mapped providing a normalized estimation of gene expression based on RNA-seq data. (B) SCope transcriptome data from the unfiltered adult fly brain dataset. (C) Relative abundance of pdzd8 transcript in controls compared with pdzd8-RNAi normalized to the relative to geometric mean of housekeeping genes αTub84B, vkg, COX8, and Rpl32; P = 0.0134. (D) Replicate of Fig 1B LacZ-RNAi with high magnification examples of MERCs (D′-D‴). Scale bar 500 nm. (E) Representative structured illumination microscopy images of ER (green) and mitochondria (purple) in L3 larval neurons. Scale bar = 5 μm.
- Figure S2. SPLICS and tether constructs used in this study.
(A) Cartoon of SPLICS targeting and mode of action (Created with BioRender.com). (B) Density of SPLICS puncta in axons is different in different axon bundles. Scale bars: fluorescence, 5 μm; light, 30 μm. (C) Cartoon of the synthetic tether construct targeting and mode of action (Created with BioRender.com). (D) SPLICS puncta indicating contact sites in larval axons. Scale bar = 5 μm. (D, E) Quantification of SPLICS puncta in (D). n = 9 larvae per genotype, three ROIs averaged per larva, unpaired t test with Welch’s Correction.
- Figure 2. Lifespan and locomotor activity changes in aged flies with pan-neuronal driven alterations in tethering.
(A, B) Locomotor activity of flies was assessed during aging by negative geotaxis climbing assays on the indicated days. n > 50 flies per genotype. Flies expressing (A) pdzd8-RNAi or (B) synthetic tether were compared with LacZ-RNAi controls. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s post hoc correction. **P < 0.01, ***P < 0.001, ****P < 0.0001. (C, D) Lifespans in standard growth conditions and food. (C) Flies expressing pdzd8-RNAi were compared with LacZ-RNAi controls. n = 97, 108 per genotype, median survival 44 versus 52 d, P < 0.0001. (D) Flies expressing the synthetic tether were compared with LacZ-RNAi controls. n = 74, 85 per genotype, median survival 52 versus 33 d, P < 0.0001.
- Figure S3. pdzd8 knockdown delays aged motor decline compared with multiple controls.
(A, B, C, D, E) Locomotor activity following pan-neuronal knockdown of pdzd8 or controls (mitoGFP, Luciferase-RNAi or LacZ-RNAi) at 2 d (A), 10 d (B), 20 d (C), 30 d (D), and 40 d (E) post-eclosion. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s post hoc correction. **P < 0.01, ***P < 0.001, ****P < 0.0001.
- Figure S4. Phenotypic characterization of altered tethering in motor neurons.
(A) Motor neuron-specific aged climbing assay showing pdzd8-RNAi compared with LacZ-RNAi controls. (B) pdzd8-RNAi rescues aged climbing defect resulting from pdzd8 overexpression, rescued by co-expression with pdzd8-RNAi. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s post hoc correction. (C) ATP levels in fly heads normalized to total protein content and show mean ± SD, n = 3, 40 flies per replicate, compared using a two-tailed t test, all differences ns. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) OK371>tether expression resulted in severely deformed NMJs on muscle 4 and made it impossible to distinguish NMJs type 1s and 1b synaptic boutons (therefore, mitochondrial density could not be quantified).
- Figure 3. Knockdown of pdzd8 protects flies against mitochondrial toxins.
Lifespans of flies expressing pan-neuronal pdzd8-RNAi were compared with LacZ-RNAi controls when aged on a restricted diet of food containing 1% agar with 5% sucrose. (A) Lifespan with dietary restriction alone. N = 62 versus 64, median survival 20 versus 19 d, difference ns. (B) Lifespan with addition of 5% hydrogen peroxide. Median survival: 63 versus 74 h, n = 67, 74, P = 0.0002. (C) Lifespan with addition of 1 mM rotenone. Median survival: 11 versus 20 d, n = 66, 57, P < 0.0001. (D) Lifespan with addition of 5 μg/ml antimycin A. Median survival: 74 versus 68 h, n = 72, 68, P = 0.0002.
- Figure 4. Knockdown of pdzd8 in larval neurons causes minor defects, whereas increasing MERCs is detrimental in axonal mitochondria size and motility.
(A) Representative images of mitochondrial morphology and distribution in larval axons. Mitochondria were detected using mitoGFP in controls (LacZ-RNAi) and pdzd8-RNAi expressing larvae. Scale bar = 5 μm. (A, B, C) Mitochondrial length (B), and mitochondrial density (C) in the larval axons shown in (A) were analysed using ordinary one-way ANOVA and Holm–Sidak’s multiple comparisons. n = 10, 10, 13 animals, data points represent different axons, all differences ns. (D) Representative kymographs showing motility of mitoGFP signal in controls and pdzd8-RNAi–expressing larvae. Stationary mitochondria appear as vertical lines, moving mitochondria form diagonal lines in anterograde or retrograde directions. (D, E) Quantification of mitochondrial transport shown in (D), analysed using ordinary one-way ANOVA and Holm–Sidak’s multiple comparisons, n = 14-25 larvae, P < 0.0001. (F) Representative images of NMJs and mitochondria of controls and pdzd8-RNAi labeled using mitoGFP. magenta = mitoGFP, green = anti-HRP (neuronal membrane). Scale bar = 10 μm. (G, H, I) Quantifications of total volume, P = 0.0036, (G), mitochondrial volume, P = 0.002 (H) and mitochondrial density (I) of 16–20 NMJs were compared using an unpaired t test with Welch’s Correction.
- Figure 5. Pan-neuronal pdzd8-RNAi increases mitophagy during aging.
(A) Representative images of MitoQC signal in wandering L3 larval ventral ganglia. magenta = mCherry, green = GFP, images show a single plane of a Z stack. Scale bar = 2 μm. (A, B) Quantification of MitoQC puncta shown in (A), n = 9 ROIs, differences ns. (C) Representative images of MitoQC signal in adult brains in 2- and 20-d-old flies, magenta = mCherry, green = GFP. Scale bar = 5 μm, image shows a single plane of a Z stack. (D, E) Quantification of MitoQC signal in adult brains and compared using an unpaired t test with Welch’s correction (n = 7–9 ROIs, P = 0.0072) (E) the Representative images of MitoQC signal in 14-d-old fly wings. Only mCherry signal (magenta) is shown for clarity. Wing nerve are outlined (white). Scale bar = 5 μm. (F) Quantification of MitoQC signal in aged fly wings at 2, 14, and 30 d post-eclosion using a one-way ANOVA with Holm–Sidak’s multiple comparisons. n (2 d) = 33, 26 wings (14 d) = 24, 31 wings (30 d) = 32, 12 wings. **P < 0.01, ***P < 0.001, ****P < 0.0001.
- Figure 6. Reducing pdzd8-mediated MERCs rescues the locomotor defects in an Alzheimer’s disease model.
(A) SPLICS puncta indicating MERCs in axon bundles of larval neurons from Aβ42 expressing flies compared with controls. Quantified puncta highlighted with V. (A, B) Quantification of SPLICS signal in (A) using Kruskal–Wallis test with Dunn’s post hoc correction, n = 6 larvae per genotype, three ROIs averaged per larva. (C) Locomotor (climbing) activity of flies of the indicated ages, comparing control versus Aβ42 with control or pdzd8-RNAi. n > 65 flies. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s post hoc correction. (D) Quantification of MitoQC signal in aged fly wings at 2 and 10 d post-eclosion using Kruskal–Wallis test with Dunn’s post hoc correction, n = 14 wings. (E, F, G) Representative recordings of mitochondrial calcium retention capacity, monitoring extramitochondrial Calcium-Green 5N fluorescence levels (AU), of mitochondria from flies of the indicated genotypes. Arrows indicate calcium pulse where retention capacity is exceeded. (E, H) Quantification of calcium retention capacity in (E). n = 3, one-way ANOVA with Dunnett’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Materials
Table S2 Genotypes in figures.
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