Prefoldin 5 is a microtubule-associated protein that suppresses Tau aggregation and neurotoxicity
- Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, India
- Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, India
- Trinity College Institute of Neuroscience (TCIN), University of Dublin, Ireland
Figures
Figure 1 with 2 supplements
Prefoldin cochaperones are genetic modifiers of Tubulin-associated unit (Tau)-induced eye degeneration.
(A–L) Bright-field images of 7-day-old Drosophila eyes expressing (A) hTauV337M (control), (B) GMR-Gal4>UAS-hTauV337M; UAS-GFP (Gal4 dilution control), (C) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn4 RNAi, (D), (E), (F) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5 RNAi, (G), (H), (I) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6 RNAi, (J) GMR-Gal4>UAS-hTauV337M; UAS-TBCE RNAi, (K) GMR-Gal4>UAS-hTauV337M; UAS-CCT5 RNAi, (L) GMR-Gal4>UAS-hTauV337M; UAS-CCT7 RNAi. (N) Histogram showing the percentage of degenerated area in eyes of 7-day-old flies of genotypes: UAS-hTauV337M/+; GMR-Gal4/+ (42.74±1.59), UAS-hTauV337M/+; GMR-Gal4/UAS-GFP (46.7±1.79), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn4 RNAi (BL77412; 63.9±2.95), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn5 RNAi (BL67815; 61.6±3.6), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn5 RNAi (KK100796; 85.06±3.01), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn5 RNAi (GD29812; 64.44±4.91), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 RNAi (BL65365; 70.95±4.17), UAS-hTauV337M/+; GMR-Gal4/+; UAS-Pfdn6 RNAi/+ (GD34204; 98.57±0.4), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 RNAi (KK101541; 77.11±2.96), UAS-hTauV337M/+; GMR-Gal4/+; UAS-TBCE RNAi/+ (BL34537; 62.01±5.65), UAS-hTauV337M/+; GMR-Gal4/+; UAS-CCT5 RNAi/+ (BL41818; 64.36±2.43), UAS-hTauV337M/+; GMR-Gal4/+; UAS-CCT7 RNAi/+ (BL34931; 61.67±4.47). *p<0.05; ***p<0.001; ns, not significant. At least 6 brightfield eye images of each genotype were used for quantification.
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Figure 1—source data 1
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Figure 1—figure supplement 1
Screening of Drosophila chaperones to identify modifiers of Tauopathy.
(A) Schematic representation of the genetic screen performed to identify the chaperones that function as modifiers of Tauopathy. In this screen, 109 RNAi lines corresponding to 64 chaperones were used, among which 15 RNAi lines against 15 genes were scored for their role as suppressors. While 20 genes (31 RNAi lines) enhanced the hTau phenotype, 46 genes (58 RNAi lines) did not alter the hTau phenotype in the eyes. Nineteen genes showed variable results with different RNAi lines, which we did not consider as either enhancers or suppressors. (B–AG) Brightfield images of 7-day-old Drosophila eyes coexpressing hTauV337M and various RNAi lines against Drosophila chaperones. (AH) Histogram showing the percentage of degenerated area in 7 day flies in coexpressing hTauV337M and various RNAi lines against chaperones. At least three brightfield eye images of each genotype were used for quantification. The details of the RNAi lines and statistical analysis are listed in Supplementary file 1.
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Figure 1—figure supplement 1—source data 1
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Figure 1—figure supplement 2
qPCR analysis for knockdown efficiency of RNAi lines against cytoskeleton regulatory chaperones.
A histogram showing the transcript levels of the mentioned genes. Total RNA was isolated from larval fillets of actin5C-Gal4 driven RNAi lines. rp49 was used as an internal control. The knockdown animals showed about 50–60% reduction in the transcript level. Three independent qRT-PCRs were performed for each genotype. Error bars represent SEM. ***p<0.001, **p<0.01.
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Figure 1—figure supplement 2—source data 1
Source data related to Figure 1—figure supplement 2.
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Figure 2 with 2 supplements
Loss of Pfdn5 disrupts microtubule organization.
(A) Generation of a loss-of-function mutant of Pfdn5 using CRISPR/Cas9-based genome editing. Schematic representation of the Pfdn5 genomic organization showing exons (solid black boxes, 1–3) and introns (thin black lines). Two loss-of-function Pfdn5 mutants with 606 bp (line-15) or 577 bp (line-40) deletion were obtained. Both mutant lines are third-instar larval lethal. (B) Schematic representation of Futsch loop organization in muscle 4 of A2 hemisegment in wild-type or Pfdn5 mutant. Pfdn5 mutant shows diffused Futsch loop organization and reduced loops at the terminal boutons. (C-E') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment showing Futsch loops in (C-C') control, (D-D') ΔPfdn515/40, (E-E') Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 double immunolabeled with neuronal membrane marker, HRP (green) and 22C10 antibody against microtubule-associated protein, Futsch (magenta). The scale bar in E' for (C-E') represents 10 µm. The inset shows a 2.0 x magnified Futsch loop. (F) Histogram showing the percentage of Futsch positive loops from muscle 4 at A2 hemi-segment in control (19.98±2.18), ΔPfdn515/40 (7.72±1.62), Elav-Gal4/+; UAS-Pfdn5/+; ∆Pfdn515/40 (27.39±2.21). ***p<0.001. At least 16 neuromuscular junctions (NMJs) of each genotype were used for quantification. (G) Western blot showing protein levels of α-Tubulin, β-Tubulin, ace-Tubulin, and Actin in control, ΔPfdn515/15, ΔPfdn540/40, ∆Pfdn15/40, and actin5C-Gal4>UAS-Pfdn5; ∆Pfdn515/40. Ran protein levels were used as an internal loading control. (H) Histogram showing the percentage of ace-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/15 (0.50±0.05), ∆Pfdn40/40 (0.49±0.04), ΔPfdn515/40 (0.46±0.04), actin5C-Gal4/UAS-Pfdn5; ∆Pfdn515/40 (1.05±0.13). **p<0.01. Three independent western blots were used for quantification. (I) Histogram showing percentage of α-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/15 (0.27±0.04), ∆Pfdn40/40 (0.16±0.02), ΔPfdn515/40 (0.11±0.04), actin5C-Gal4/UAS-Pfdn5; ∆Pfdn515/40 (0.85±0.07). ***p<0.001. Three independent western blots were used for quantification. (J) Histogram showing percentage of β-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/15 (0.52±0.04), ∆Pfdn40/40 (0.39±0.04), ΔPfdn515/40 (0.30±0.09), actin5C-Gal4/UAS-Pfdn5; ∆Pfdn515/40 (1.03±0.11). **p<0.01. Three independent western blots were used for quantification.
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Figure 2—source data 1
Source data related to Figure 2.
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Figure 2—source data 2
PDF files containing original Western blots for Figure 2G, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig2-data2-v1.zip
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Figure 2—source data 3
Original files for Western blot analysis displayed in Figure 2G.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig2-data3-v1.zip
Figure 2—figure supplement 1
Pfdn5 mutants are null alleles with no detectable Pfdn5 transcripts.
(A) Semi-quantitative RT-PCR showing transcript levels of Pfdn5 in control, ΔPfdn515/15, ΔPfdn540/40, ΔPfdn515/40, and actin5C-Gal4>UAS-Pfdn5; ∆Pfdn515/40. rp49 was used as an internal control for RT-PCR. (B–E) Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (B) control, (C) ΔPfdn515/15 (D) ΔPfdn540/40 (E) ΔPfdn515/40 double immunolabeled for HRP (green) and CSP (magenta). The insets represent the 3 X magnified portion of the image shown in a white box. The scale bar in E for (B–E) represents 10 µm. (F) Histogram showing the number of satellite boutons from muscle 4 at A2 hemisegment in control (2.63±0.59), ΔPfdn515/15 (7.0±0.7), ΔPfdn540/40 (6.63±0.7), and ΔPfdn515/40 (7.2±1.1). ***p<0.001. At least 16 NMJs of each genotype were used for quantification. (G) Histogram showing bouton area (in µm2) from muscle 4 at A2 hemisegment in control (4.2±0.3), ΔPfdn515/15 (4.9±0.2), ΔPfdn540/40 (5.0±0.2), and ΔPfdn515/40 (5.1±0.3). ns, not significant. At least 16 NMJs of each genotype were used for quantification. (H) Histogram showing the number of branches per NMJ from muscle 4 at A2 hemisegment in control (3.12±0.2), ΔPfdn515/15 (2.6±0.2), ΔPfdn540/40 (3.10±0.4), and ΔPfdn515/40 (2.6±0.3). ns, not significant. At least 16 NMJs of each genotype were used for quantification. (I) Histogram showing total bouton number from muscle 4 at A2 hemi-segment in control (35.25±1.8), ΔPfdn515/15 (36.06±2.0), ΔPfdn540/40 (39.0±2.3), and ΔPfdn515/40 (38.4±2.1). ns, not significant. At least 16 NMJs of each genotype were used for quantification.
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Figure 2—figure supplement 1—source data 1
Source data related to Figure 2—figure supplement 1.
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Figure 2—figure supplement 2
Loss of Pfdn5 disrupts microtubule cytoskeleton.
(A–C) Confocal images showing the muscle 2 of A2 hemisegment in control, ΔPfdn515/40, and mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40 double immunolabeled with ace-Tubulin (red) and Hoechst (cyan). The scale bar in the C represents 20 µm for images (A–C). (D) Histogram showing the average fluorescence intensity (au) of ace-tubulin at muscle 2 of A2 hemisegment in control (530.1±71.6), ΔPfdn515/40 (141.9±8.43), and mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40 (395.1±66.78). **p<0.01. At least 17 neuromuscular junctions (NMJs) of each genotype were used for quantification. (E-G') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment showing synaptic levels of ace-tubulin in control, ΔPfdn515/40, and Elav-Gal4>UAS-Pfdn5; ΔPfdn515/40 double immunolabeled for ace-Tubulin (magenta) and HRP (green). The scale bar in the G' represents 20 µm for images (E-G'). (H) Histogram showing average fluorescence intensity of ace-tubulin at muscle 4 NMJ in control (0.54±0.04), ΔPfdn515/40 (0.36±0.03), and Elav-Gal4>UAS-Pfdn5; ΔPfdn515/40 (0.53±0.02). **p<0.01. At least 8 NMJs of each genotype were used for quantification. (I) Semi-quantitative RT-PCR showing transcript levels of α-tubulin in control, ΔPfdn515/15, ΔPfdn540/40, ΔPfdn515/40, and actin5C-Gal4>UAS-Pfdn5; ∆Pfdn515/40. rp49 was used as an internal control.
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Figure 2—figure supplement 2—source data 1
Source data related to Figure 2—figure supplement 2.
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Figure 3 with 1 supplement
Pfdn5 is a novel microtubule-binding protein.
(A–C''') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment triple-labeled for Pfdn5 (green), α-Tubulin (blue), and HRP (magenta) showing that Pfdn5 colocalizes with microtubule cytoskeleton in (A-A''') in the wild-type larval neuronal axons and in the tracheal tubes, (B-B''') Pfdn5 mutants show dramatically reduced Pfdn5 and α-Tubulin levels, (C-C''') actin5C-Gal4-mediated rescue (actin5C-Gal4>UAS-Pfdn5; ∆Pfdn515/40) significantly restored the level of Pfdn5 and α-Tubulin. Arrows represent Pfdn5 colocalization with microtubule loops, which are not detectable in the Pfdn5 mutants. The scale bar in C''' (for A-C''') represents 10 µm. (D) Pearson’s correlation coefficient to quantify colocalization between Pfdn5 and axonal microtubule. (E) Schematic representation of the microtubule-binding protocol. Head lysate from wild-type flies, treated with Taxol or Nocodazole, was subjected to ultracentrifugation. The ‘soluble fraction’ contains free tubulin, whereas the ‘insoluble pellet fraction’ contains the stabilized microtubule along with microtubule-binding proteins, which can be detected by western blotting. The details of the protocol is described in the material and methods section. (F) Microtubule binding assay with Drosophila head lysate in the presence of Taxol or Nocodazole (diluted in DMSO). T represents (Total fraction: input fraction), S represents (Supernatant: free tubulin), and P represents (Pellet fraction: stabilized microtubule). Immunoblot with antibodies against ace-Tubulin or Pfdn5 detected increased Pfdn5 in the pellet fraction in the presence of Taxol but not Nocodazole. The binding of Pfdn5 with stabilized microtubules was calculated as the percentage of Pfdn5 in the pellet fraction. Ran was used as the loading control. (G) Histogram showing the percentage of the Pfdn5 in the pellet fraction of in vivo microtubule binding assay in the presence of DMSO (7.83±2.92), Nocodazole (0.42±0.1), or Taxol (36.67±7.56). Five independent western blots were used for quantification.
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Figure 3—source data 1
Source data related to Figure 3.
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Figure 3—source data 2
PDF files containing original Western blots for Figure 3F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig3-data2-v1.zip
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Figure 3—source data 3
Original files for Western blot analysis displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig3-data3-v1.zip
Figure 3—figure supplement 1
Generation and characterization of Pfdn5 antibody.
(A) Western blot showing the levels of Pfdn5 in control, ΔPfdn515/40, and actin5c-Gal4>UAS-Pfdn5; ΔPfdn515/40. Ran was used as a loading control. (B-D') Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing Pfdn5 levels in (C-C') control (D-D') ΔPfdn515/40 (E-E') actin5c-Gal4>UAS-Pfdn5; ΔPfdn515/40 double immunolabeled for HRP (magenta) and Pfdn5 (green). The scale bar in D' for (B-D') represents 10 µm. (E) Histogram showing the average fluorescence intensity of Pfdn5 at the NMJ of muscle 4 in control (0.41±0.02), ΔPfdn515/40 (0.15±0.01), and actin5c-Gal4>UAS-Pfdn5; ΔPfdn515/40 (0.46±0.02). ***p<0.001; ns, not significant. At least eight NMJs of each genotype were used for quantification. (F) Western blot showing the levels of Pfdn5 and Tubulin in control, ΔPfdn515/+, and ΔPfdn540/+. GAPDH was used as a loading control for Pfdn5. Ran was used as a loading control for Tubulin. (G) Histogram showing the level of Pfdn5 normalized with GAPDH in control (1.00±0.00), ΔPfdn515/+ (0.81±0.05), and ΔPfdn540/+ (0.89±0.09). ns, not significant. (H) Histogram showing the level of Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/+ (0.87±0.07), and ΔPfdn540/+ (0.95±0.10). ns, not significant.
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Figure 3—figure supplement 1—source data 1
Source data related to Figure 3—figure supplement 1.
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Figure 3—figure supplement 1—source data 2
PDF files containing original Western blots for Figure 3—figure supplement 1A and F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig3-figsupp1-data2-v1.zip
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Figure 3—figure supplement 1—source data 3
Original files for Western blot analysis displayed in Figure 3—figure supplement 1A and F.
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Figure 4 with 1 supplement
Loss of Pfdn5 mimics and enhances Tubulin-associated unit (Tau)-induced synaptic defects.
(A–H') Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (A-A') control, (B-B') ΔPfdn515/40, (C-C') Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40, (D-D') mef2-Gal4>UAS-Pfdn5; ∆Pfdn515/40, (E-E') Elav-Gal4/+ (Gal4 control), (F-F') Elav-Gal4>UAS-hTauV337M, (G-G') Elav-Gal4>hTauVV337M; ∆Pfdn515/40, (H-H') Elav-Gal4>hTauVV337M;UAS-Pfdn5; ∆Pfdn515/40 double immunolabeled for HRP (green), and CSP (magenta). The scale bar in H for (A-H') represents 10 µm. Arrows point to clustered satellite boutons. (I) Histogram showing total number of boutons from muscle 4 at A2 hemisegment in control (35.25±1.8), ΔPfdn515/40 (38.38±2.15), Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (31.94±1.18), mef2-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (38.40±2.17), Elav-Gal4/+ (28.13±1.51), Elav-Gal4>UAS-hTauV337M (36.00±2.65), Elav-Gal4>hTauVV337M; ∆Pfdn515/40 (60.34±3.76), Elav-Gal4>hTauVV337M; UAS-Pfdn5; ∆Pfdn515/40 (33.69±1.76). ***p<0.001; ns, not significant. At least 12 NMJs of each genotype were used for quantification. (J) Histogram showing number of satellite boutons from muscle 4 at A2 hemisegment in control (2.25±0.41), ΔPfdn515/40 (18.25±1.28), Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (2.94±0.67), mef2-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (15.6±0.86), Elav-Gal4/+ (2.2±0.38), Elav-Gal4>UAS-hTauV337M (14.06±1.00), Elav-Gal4>hTauVV337M; ∆Pfdn515/40 (32.25±3.2), Elav-Gal4>hTauVV337M;UAS-Pfdn5; ∆Pfdn515/40 (4.0±0.5). ***p<0.001; ns, not significant. At least 12 NMJs of each genotype were used for quantification. (K) Histogram showing bouton area from muscle 4 at A2 hemi segment in control (5.7±0.29), ΔPfdn515/40 (5.1±0.28), Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (5.6±0.2), mef2-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (5.8±0.2), Elav-Gal4/+ (5.2±0.4), Elav-Gal4>UAS-hTauV337M (6.4±0.4), Elav-Gal4>hTauVV337M; ∆Pfdn515/40 (2.3±0.2), Elav-Gal4>hTauVV337M;UAS-Pfdn5; ∆Pfdn515/40 (6.3±0.3). ***p<0.001; ns, not significant. At least 12 NMJ of each genotype were used for quantification.
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Figure 4—source data 1
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Figure 4—figure supplement 1
Loss of Pfdn5 enhances hTauR406W-induced synaptic phenotypes.
(A–C) Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing synaptic morphology in control, Elav-Gal4>hTauRR406W, and Elav-Gal4>hTauRR406W; ΔPfdn515/40 double immunolabeled for CSP (magenta) and HRP (green). The inset shows the magnified images of the NMJs. The scale bar in the C represents 10 µm for images (A–C). (D) Histogram showing total bouton number from muscle 4 at A2 hemisegment in control (32.08±2.14), ΔPfdn515/40 (36.08±2.50), Elav-Gal/+ (26.13±1.58), Elav-Gal4>hTauRR406W (33.38±1.97), and Elav-Gal4>hTauRR406W; ΔPfdn515/40 (57.87±4.32). ***p<0.001; ns, not significant. At least 13 NMJs of each genotype were used for quantification. (E) Histogram showing the average bouton area from muscle 4 at A2 hemisegment in control (5.15±0.18), ΔPfdn515/40 (6.29±0.62), Elav-Gal/+ (5.15±0.15), Elav-Gal4>hTauRR406W (6.75±0.43), and Elav-Gal4>hTauRR406W; ΔPfdn515/40 (2.0±0.14). ***p<0.001; ns, not significant. At least 13 NMJs of each genotype were used for quantification. (F-I') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment showing futsch intensity in control (F-F'), Elav-Gal4>hTauVV337M (G-G'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (H-H') and Elav-Gal4>hTauVV337M; UAS-Pfdn5 (I-I') double immunolabeled for 22C10 (magenta) and HRP (green). The scale bar in the I' represents 10 µm for images (F-I'). (J) Histogram showing futsch intensity normalized with HRP from muscle 4 at A2 hemisegment in control (62.18±2.56), ΔPfdn515/40 (48.32±2.66), Elav-Gal4>hTauVV337M (48.75±1.79), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (31.58±2.0), and Elav-Gal4>hTauVV337M; UAS-Pfdn5 (68.94±3.78). **p<0.01; ***p<0.001; ns, not significant. At least 13 NMJs of each genotype were used for quantification.
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Figure 4—figure supplement 1—source data 1
Source data related to Figure 4—figure supplement 1.
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Figure 5 with 3 supplements
Loss of Pfdn5 induces formation of hTauV337M aggregates in larval neurons.
(A) Schematic representation of pathological hTau distribution in larval brain lobes and axons of the control (left half) or Pfdn5 mutant (right half) animals. (B–E') Confocal single-section images of third instar larval brain in (B-B') Elav-Gal4/+ (control), (C-C') Elav-Gal4>UAS-hTauV337M, (D-D') Elav-Gal4>UAS-hTauV337M; ∆Pfdn515/40, (E-E') Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5; ∆Pfdn515/40 double immunolabeled with neuronal membrane marker, HRP (magenta), and T46 antibody against hTau (green). The scale bar in E' for (B-E') represents 10 µm. Arrows in D and D' point to the hTau punctae/flame-shaped aggregates in the brain. (F) Histogram showing the quantification of the number of hTau punctae (>3 μm2) in Elav-Gal4>UAS-hTauV337M (1.13±0.39), Elav-Gal4>UAS-hTauV337M; ΔPfdn515/40 (10.5±2.57), and Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5/+; ΔPfdn515/40 (0.17±0.17). ***p<0.001. At least six optic lobes of each genotype were used for quantification. (G-J'') Confocal single section images of third instar larval axons in (G-G'') Elav-Gal4/+ (control), (H-H'') Elav-Gal4>UAS-hTauV337M, (I-I'') Elav-Gal4>UAS-hTauV337M; ∆Pfdn515/40 (J-J'') Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5; ∆Pfdn515/40 double immunolabeled for HRP (magenta), and T46 antibody against hTau (green). The scale bar in J'' for (G-J'') represents 10 µm. Arrows in I and I'' point to the hTauV337M aggregates in axons. (K) Histogram showing the quantification of the number of hTau punctae normalized with HRP positive area in Elav-Gal4>UAS-hTauV337M (0.25±0.1), Elav-Gal4>UAS-hTauV337M; ΔPfdn515/40 (2.96±0.4), and Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5/+; ΔPfdn515/40 (0.29±0.1). ***p<0.001. At least 20 axons from eight animals of each genotype were used for quantification. (L) Histogram showing the intensity of total hTau normalized with HRP in Elav-Gal4/UAS-hTauV337M (0.59±0.02), Elav-Gal4>UAS-hTauV337M; ΔPfdn515/40 (0.27±0.02), and Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5/+; ΔPfdn515/40 (0.46±0.4). ***p<0.001. At least 20 axons from eight animals of each genotype were used for quantification.
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Figure 5—source data 1
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Figure 5—figure supplement 1
hTau aggregates in the axons and larval brain of the Pfdn5 mutants.
(A–B) Confocal images showing the intensity profile across the axons labeled with the T46 (total Tubulin-associated unit, Tau) in Elav-Gal4>hTauVV337M and Elav-Gal4>hTauVV337M; ΔPfdn515/40. The line drawn across the axons is 6 µm, and the scale bar in B represents 5 µm for (A–B). (C) Intensity plot profile showing the intensity and distribution of the T46 across the axons in the Elav-Gal4>hTauVV337M (green) and Elav-Gal4>hTauVV337M; ΔPfdn515/40 (magenta). (D-E'') Confocal single-section images of third instar larval axons in (D-D'') Elav-Gal4>UAS-hTauV337M, (E-E'') Elav-Gal4>UAS-hTauV337M; Pfdn515/40 double immunolabeled with neuronal membrane marker, HRP (magenta), and AT8 antibody against phospho-Tau (green). The scale bar in E'' for (D-E'') represents 10 µm. Arrows in (E) point to the punctae or the aggregates of Tau. (F) Histogram showing the quantification of the number of phospho-Tau punctae normalized with HRP positive area in Elav-Gal4>UAS-hTauV337M (0.002±0.0005), Elav-Gal4>UAS-hTauV337M; ΔPfdn515/40 (0.03±0.006). ***p<0.001; ns, not significant. At least 36 axons from eight animals of each genotype were used for quantification. (G-H') Confocal images of third instar larval brain showing hTau aggregates in Elav-Gal4>UAS-hTauWT (G-G'), Elav-Gal4>UAS-hTauWT; Pfdn515/40 (H-H') double immunolabeled with neuronal membrane marker, HRP (magenta), and T46 antibody against total Tau (green). The scale bar in H' for (G-H') represents 20 µm. (I) Histogram showing the quantification of the number of total Tau punctae (>3 μm2) per lobe in Elav-Gal4>UAS-hTauWT (0.14±0.14), Elav-Gal4>UAS-hTauWT; ΔPfdn515/40 (15±1.09). ***p<0.001; ns, not significant. At least seven lobes of each genotype were used for quantification.
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Figure 5—figure supplement 1—source data 1
Source data related to Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
Loss of Pfdn5 results in the formation of stable hTau aggregates.
(A) Western blot showing the Tubulin-associated unit (Tau) protein level in supernatant and pellet fraction of Elav-Gal4>hTauVV337M and Elav-Gal4>hTauVV337M; ΔPfdn515/40. GAPDH was used as a loading control. (B) Histogram showing the level of Tau in supernatant fraction normalized with GAPDH in Elav-Gal4>hTauVV337M (1.00±0.00), and Elav-Gal4>hTauVV337M; ΔPfdn515/40 (0.52±0.17). *p<0.05; ns, not significant. (C) Histogram showing the level of Tau in pellet fraction normalized in Elav-Gal4>hTauVV337M (1.00±0.00), and Elav-Gal4>hTauVV337M; ΔPfdn515/40 (1.25±0.03). **p<0.01; ns, not significant. (D-G') Confocal images of third instar larval brain showing hTau aggregates in Elav-Gal4>hTauVV337M with 1% 1,6-Hexanediol (D-D'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 1% 1,6-Hexanediol (E-E'), Elav-Gal4>hTauVV337M with 5% 1,6-Hexanediol (F-F'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 5% 1,6-Hexanediol (G-G') double immunolabeled with neuronal membrane marker, HRP (magenta), and T46 antibody against total Tau (green). The scale bar in G' for (D-G') represents 10 µm. (H-K'') Confocal images of third instar larval axons showing hTau aggregates in Elav-Gal4>hTauVV337M with 1% 1,6-Hexanediol (H-H''), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 1% 1,6-Hexanediol (I-I''), Elav-Gal4>hTauVV337M with 5% 1,6-Hexanediol (J-J''), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 5% 1,6-Hexanediol (K-K'') double immunolabeled with neuronal membrane marker, HRP (magenta), and T46 antibody against total Tau (green). The scale bar in K'' for (H-K'') represents 10 µm. (L) Histogram showing the quantification of the number of total Tau per 100 μm2 of HRP positive area in Elav-Gal4>hTauVV337M with 0% 1,6-Hexanediol (0.28±0.12), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 0% 1,6-Hexanediol (3.35±0.45), Elav-Gal4>hTauVV337M with 1% 1,6-Hexanediol (0.21±0.04), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 1% 1,6-Hexanediol (2.47±0.21), Elav-Gal4>hTauVV337M with 5% 1,6-Hexanediol (0.15±0.04), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with 5% 1,6-Hexanediol (2.39±0.18). *p<0.05; ***p<0.001; ns, not significant. At least 24 axons from eight animals of each genotype were used for quantification. (M-N') Confocal images of third instar larval brain showing localization of hTau aggregates in Elav-Gal4>hTauVV337M (M-M'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (N-N') double immunolabeled with Elav (magenta), and T46 antibody against total Tau (green). The scale bar in N' for (M-N') represents 10 µm.
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Figure 5—figure supplement 2—source data 1
Source data related to Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig5-figsupp2-data1-v1.xlsx
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Figure 5—figure supplement 2—source data 2
PDF files containing original Western blots for Figure 5—figure supplement 2A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig5-figsupp2-data2-v1.zip
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Figure 5—figure supplement 2—source data 3
Original files for Western blot analysis displayed in Figure 5—figure supplement 2A.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig5-figsupp2-data3-v1.zip
Figure 5—figure supplement 3
Loss of Pfdn5 induces Tau aggregation independent of hyperphosphorylation.
(A-D''') Confocal images of third instar larval brain showing hTau aggregates in Elav-Gal4>hTauWT (A-A'''), Elav-Gal4>hTauWT; ΔPfdn515/40 (B-B'''), Elav-Gal4>hTauVV337M (C-C'''), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (D-D''') triple immunolabeled with neuronal membrane marker, HRP (red), AT8 antibody against p-Tau (blue) and D5D8N antibody against total Tubulin-associated unit (Tau) (green). The scale bar in D''' for (A-D''') represents 20 µm. The arrow points to the mutually exclusive Tau aggregates. (E) Histogram showing the quantification of the number of Tau punctae (>3 μm2) per lobe in Elav-Gal>UAS-hTauWT; ΔPfdn515/40 and Elav-Gal4>UAS-hTauV337M; ΔPfdn515/40. (F-I') Confocal images of third instar larval brain showing hTau aggregates in Elav-Gal4>hTauVV337M (F-F'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (G-G') without LiCl, Elav-Gal4>hTauVV337M (H-H'), Elav-Gal4>hTauVV337M; ΔPfdn515/40 (I-I') in the presence of 20 mM double immunolabeled with neuronal membrane marker, HRP (magenta), and T46 antibody against total Tau (green). The scale bar in I' for (F-I') represents 10 µm. (J) Histogram showing the quantification of the number of total Tau punctae (>3 μm2) per lobe in Elav-Gal4>hTauVV337M without LiCl (0.67±0.33), Elav-Gal4>hTauVV337M with LiCl (0.67±0.49), Elav-Gal4>hTauVV337M; ΔPfdn515/40 without LiCl (17.5±1.61), Elav-Gal4>hTauVV337M; ΔPfdn515/40 with LiCl (19.51±1.32). ***p<0.001; ns, not significant. At least six lobes of each genotype were used for quantification.
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Figure 5—figure supplement 3—source data 1
Source data related to Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig5-figsupp3-data1-v1.xlsx
Figure 6 with 3 supplements
Overexpression of Pfdn5 or Pfdn6 suppresses age-dependent progression of hTau-induced neurodegeneration.
(A–D) Bright-field images of Drosophila eyes expressing (A) GMR-Gal4/+ (control), (B) GMR-Gal4>UAS-hTauV337M, (C) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (D) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. (A'–D') Scanning electron microscopic images of Drosophila eyes expressing (A') GMR-Gal4/+ (control), (B') GMR-Gal4>UAS-hTauV337M, (C') GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (D') GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. (E) Histogram showing the percentage of fused ommatidia in GMR-Gal4/+ (0.00±0.00), UAS-hTauV337M/+; GMR-Gal4/+ (29.12±2.37), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn5 (2.98±0.31), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 (2.78±0.60). ***p<0.001. At least 12 SEM eye images of each genotype were used for quantification. (F) Histogram showing percentage of degenerated area in GMR-Gal4/+ (0.00±0.00), UAS-hTauV337M/+; GMR-Gal4/+ (70.96±3.00), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn5 (5.30±0.94), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 (3.83±1.37). ***p<0.001. At least 12 SEM eye images of each genotype were used for quantification. (H–L) Confocal images of a single section of 30-day-old adult brain in (H) Elav-Gal4/+ (control), (I) Elav-Gal4>UAS-hTauV337M, (J) Elav-Gal4>hTauVV337M; UAS-GFP, (K) Elav-Gal4>hTauVV337M; UAS-Pfdn5, (L) Elav-Gal4>hTauVV337M; UAS-Pfdn6 double immunolabeled with Hoechst (cyan), and Phalloidin (magenta). The insets represent the 3 x magnified portion of the image. Arrows point to the pathological vacuolar structures. The scale bar in L for (H–L) represents 20 µm. (M) Histogram showing the quantification of number of vacuoles in 30-day-old adult brain in Elav-Gal4/+ (7.13±0.58), Elav-Gal4>UAS-hTauV337M (75.17±10.47), Elav-Gal4>hTauVV337M; UAS-GFP/+ (61.17±6.91), Elav-Gal4>hTauVV337M; UAS-Pfdn5/+ (7.86±0.91), Elav-Gal4>hTauVV337M; UAS-Pfdn6/+ (5.67±1.69). ***p<0.001. At least six brains of each genotype were used for quantification. (N) Histogram showing the quantification of vacuole size (in µm2) in 30-day-old adult brain in Elav-Gal4/+ (5.9±0.92), Elav-Gal4>UAS-hTauV337M (50.06±11.52), Elav-Gal4>hTauVV337M; UAS-GFP/+ (47.17±5.39), Elav-Gal4>hTauVV337M; UAS-Pfdn5/+(6.76±1.57), Elav-Gal4>hTauVV337M; UAS-Pfdn6/+ (4.36±1.08). ***p<0.001. At least 6 brains of each genotype were used for quantification. (O) Western blot showing protein levels of total Tau in Elav-Gal4>UAS-hTauV337M, Elav-Gal4>hTauVV337M; UAS-GFP (Gal4-dilution control), Elav-Gal4>hTauVV337M; UAS-Pfdn5, Elav-Gal4>hTauVV337M; UAS-Pfdn6. Ran protein levels were used as an internal loading control.
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Figure 6—source data 1
Source data related to Figure 6.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-data1-v1.xlsx
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Figure 6—source data 2
PDF files containing original Western blots for Figure 6O, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-data2-v1.zip
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Figure 6—source data 3
Original files for Western blot analysis displayed in Figure 6O.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-data3-v1.zip
Figure 6—figure supplement 1
Pfdn5 rescues progressive eye degeneration induced by expression of TauV337M.
(A–G) Bright-field images of 7 and 14-day-old Drosophila eyes expressing (A) GMR-Gal4/+ (control), (B and E) GMR-Gal4>UAS-hTauV337M, (C and F) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (D and G) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. (A'–G') Scanning electron microscopic images of 7 and 14-day-old Drosophila eyes expressing (A') GMR-Gal4/+ (control), (B' and E') GMR-Gal4>UAS-hTauV337M, (C' and F') GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (D' and G') GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. The scale bar in G' for (A-G') represents 100 µm. (H–I) Graph showing quantifications of age-dependent progression of ommatidial fusion (H) and percentage of degenerated eye area (I) in GMR-Gal4>UAS-hTauV337M, GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5 and GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. Note that the expression of Pfdn5 or Pfdn6 suppresses the Tubulin-associated unit (Tau)-induced progressive eye degeneration. At least 12 SEM eye images of each genotype were used for quantification at each time point.
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Figure 6—figure supplement 1—source data 1
Source data related to Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Coexpression of Pfdn5 with hTau variants rescues the ommatidial degeneration and synaptic defects.
(A–B) Bright-field images of 30-day-old Drosophila eyes expressing (A) GMR-Gal4>UAS-hTauV337M, and (B) GMR-Gal4>UAS-hTauV337M; UAS-GFP. (C–D) Bright-field images of 5-day-old Drosophila eyes expressing (E) GMR-Gal4>UAS-hTauR406W, and (F) GMR-Gal4>UAS-hTauR406W; UAS-Pfdn5. (E) Histogram showing the percentage of degenerated area in GMR-Gal4>UAS-hTauV337M (42.74±7.6), GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5 (30.03±1.74), GMR-Gal4>UAS-hTauR406W (82.15±3.19), and GMR-Gal4>UAS-hTauR406W; UAS-Pfdn5 (63.11±3.49). At least four brightfield eye images of each genotype were used for quantification. (F-J') Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (F) Elav-Gal4/+ (control), (G) Elav-Gal4>UAS-hTauWT, (H) Elav-Gal4>hTauWT; UAS-Pfdn5, (I) Elav-Gal4>hTauVV337M, (J) Elav-Gal4>hTauVV337M; UAS-Pfdn5 double immunolabeled with Hoechst (cyan), and Phalloidin (magenta). Arrows in (F) point to the pathological vacuolar structures. The scale bar in J for (F-J') represents 10 µm. (K) Histogram showing the number of boutons from muscle 4 at A2 hemisegment in Elav-Gal4/+ (control) (G), Elav-Gal4>UAS-hTauWT (H), Elav-Gal4>hTauWT; UAS-Pfdn5 (I), Elav-Gal4>hTauVV337M (J), Elav-Gal4>hTauVV337M; UAS-Pfdn5. ns; not significant. At least 16 NMJs of each genotype were used for quantification. (L) Histogram showing the number of satellite boutons from muscle 4 at A2 hemisegment in Elav-Gal4/+ (control) (G), Elav-Gal4>UAS-hTauWT (H), Elav-Gal4>hTauWT; UAS-Pfdn5 (I), Elav-Gal4>hTauVV337M (J), Elav-Gal4>hTauVV337M; UAS-Pfdn5. ***p<0.001. At least 16 NMJs of each genotype were used for quantification.
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Figure 6—figure supplement 2—source data 1
Source data related to Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-figsupp2-data1-v1.xlsx
Figure 6—figure supplement 3
Overexpression of Pfdn5 suppresses the age-dependent vacuolization in hTauV337M-expressing flies.
(A–D) Confocal images of a single section of a 2-day-old adult brain in (A) Elav-Gal4/+ (control), (B) Elav-Gal4>UAS-hTauV337M, (C) Elav-Gal4>hTauVV337M; UAS-Pfdn5, (D) Elav-Gal4>hTauVV337M; UAS-Pfdn6 double immunolabeled with Hoechst (cyan), and Phalloidin (magenta). (E–H) Confocal images of a single section of 14-day-old adult brain in (E) Elav-Gal4/+ (control), (F) Elav-Gal4>UAS-hTauV337M, (G) Elav-Gal4>hTauVV337M; UAS-Pfdn5, (H) Elav-Gal4>hTauVV337M; UAS-Pfdn6 double immunolabeled with Hoechst (cyan), and Phalloidin (magenta). Arrows in (F) point to the pathological vacuolar structures. The scale bar in H for (A–D) represents 20 µm. (I) Histogram showing the quantification of number of vacuoles in 14 days old adult brain in Elav-Gal4/+ (0.00±0.00), Elav-Gal4>UAS-hTauV337M (4.29±0.83), Elav-Gal4>hTauVV337M; UAS-GFP (3.22±0.66), Elav-Gal4>hTauVV337M; UAS-Pfdn5 (0.33±0.24), Elav-Gal4>hTauVV337M; UAS-Pfdn6 (0.44±0.24). ***p<0.001. At least 7 brains of each genotype were used for quantification. (J) Histogram showing the quantification of vacuole size (in µm2) in 14-day-old adult brain in Elav-Gal4/+ (0.00±0.00), Elav-Gal4>UAS-hTauV337M (12.36±2.40), Elav-Gal4>hTauVV337M; UAS-GFP (11.39±3.57), Elav-Gal4>hTauVV337M; UAS-Pfdn5 (0.77±0.53), Elav-Gal4>hTauVV337M; UAS-Pfdn6 (2.58±1.35). ***p<0.001. At least seven brains of each genotype were used for quantification.
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Figure 6—figure supplement 3—source data 1
Source data related to Figure 6—figure supplement 3.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig6-figsupp3-data1-v1.xlsx
Figure 7
Overexpression of Pfdn5 or Pfdn6 rescues Tubulin-associated unit (Tau)-induced defects in learning and memory.
(A) Cartoon of the Y-maze assay used for behavioural testing of conditioned odor preferences. Schematics of the protocol used to induce and measure a form of learning and memory (Mohandasan et al., 2022). During training, flies are exposed to a normally attractive odorant 2,3-butanedione (10–3-fold dilution) in a spaced training protocol: 8 X repeats of a training trial involving 5 min in the presence of unpleasant medium (80 mM CuSO4 +85 mM sucrose and 0.75% agar) followed by 5 min in an air-filled empty vial. Trained flies were tested in a binary odor-choice assay in a Y-maze apparatus for their odor vs. air preference. Control flies were trained in 0.75% agar media with 85 mM sucrose and similarly tested in Y-maze. (B–G) Histogram showing the quantification of odor preference index of naïve and trained flies towards 2,3-BD in Y-maze showing normal memory in control. A reduction in the preference index after training reflects levels of learning and memory (B) UAS-hTauV337M/+; naïve flies (23.66±2.42), trained flies (10.4±2.14), whereas pan-neuronal expression of the pathological variant hTau causes a defect in long-term memory response (C) Elav-Gal4/UAS-hTauV337M; naïve flies (28.39±3.47), trained flies (29.13±4.65). Notably, pan-neuronal overexpression of Pfdn5 (D) Elav-Gal4/+; UAS-Pfdn5/+; naïve flies (18.27±2.75), trained flies (–4.43±3.21), pan-neuronal overexpression of Pfdn6 (E) Elav-Gal4/+; UAS-Pfdn6/+; naïve flies (18.37±3.19), trained flies (–0.88±4.73) were normal. Interestingly, pan-neuronal overexpression of Pfdn5 along with hTauV337M expression rescues the learning and memory deficits in (F) Elav-Gal4/UAS-hTauV337M; UAS-Pfdn5/+; naïve flies (14.28±1.96), trained flies (–1.5±1.7). Consistently, pan-neuronal over-expression of Pfdn6 along with hTauV337M expression also rescues the learning and memory deficits in (G) Elav-Gal4/UAS-hTauV337M; UAS-Pfdn6/+; naïve flies (20.73±4.58), trained flies (–0.52±5.07). n=8 biological replicates in each case. Error bars represent the standard error of the mean (SEM). **p<0.01; ***p<0.001; ns, not significant. (H) Confocal image of Drosophila brain labeled with anti-FasII antibody (magenta) and Hoechst (cyan) showing mushroom body structure in wild-type animals. Scale bar 50 μm. (H') Confocal image of Drosophila mushroom body showing α-lobe, β-lobe, and γ-lobe. (I-M') Confocal images showing mushroom body organization in (I-I') control, (J-J') Elav-Gal4>UAS-hTauV337M Grade I (Grade I represents the defective mushroom body with one α-lobe missing), (K-K') Elav-Gal4>UAS-hTauV337M Grade II (Grade II represents the severe defects in mushroom body with both α-lobe missing), (L-L') Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5, (M-M') Elav-Gal4>UAS-hTauV337M; UAS-Pfdn6 double immunolabeled with FasII (magenta) and Hoechst (cyan). Scale bar in (M') for (I-M') represents 20 μm. The arrow points towards the β-lobe crossing midline, the arrowhead points to the thinner α lobe, and the asterisk represents the missing lobe. (N) Histogram showing the quantification for the percentage of Drosophila brain having defective mushroom body in control (0.00±0.00), Elav-Gal4>UAS-hTauV337M (91.67±8.33), Elav-Gal4>UAS-hTauV337M; UAS-GFP (93.75±6.25), Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5 (0.00±0.00), Elav-Gal4>UAS-hTauV337M; UAS-Pfdn6 (18.75±6.25). ***p<0.001; ns, not significant. The error bar represents the standard error of the mean (SEM); the statistical analysis was done using one-way ANOVA. (O) Histogram showing the quantification of mushroom body organization as normal mushroom bodies (NBs), Grade I, or Grade II. In controls (NBs: 100, Grade I: 0, Grade II: 0), Elav-Gal4>UAS-hTauV337M (NBs: 11.1, Grade I: 22.2%, Grade II: 66.7), Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5 (NBs: 100, Grade I: 0, Grade II: 0), Elav-Gal4>UAS-hTauV337M; UAS-Pfdn6 (NBs: 83.3, Grade I: 16.6, Grade II: 0).
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Figure 7—source data 1
Source data related to Figure 7.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig7-data1-v1.xlsx
Figure 8 with 1 supplement
Microtubule stability and Tubulin-associated unit (Tau) association with microtubules require Pfdn5 functions downstream of tubulin monomer expression.
(A) Western blot showing protein levels of ace-Tubulin, α-Tubulin, and β-Tubulin in control, ∆Pfdn15/40, and Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40. Ran protein levels were used as an internal loading control. (B-D') Confocal images of neuromuscular junction (NMJ) synapses showing synaptic microtubules in (B-B') control, (C-C') ΔPfdn515/40, (D-D') Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 double immunolabeled for ace-Tubulin (magenta) and HRP (green). The scale bar in D' for (A-D') represents 10 µm. Arrows in C and D show disrupted microtubules. (E) Histogram showing ace-Tubulin intensity at the NMJ in control (0.47±0.03), ΔPfdn515/40 (0.18±0.01), Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (0.23±0.01). ***p<0.001; ns, not significant. At least six NMJs of each genotype were used for quantification. (F-H') Confocal images of third instar larval axons in (F-F') Elav-Gal4>UAS-hTauV337M, (G-G') Elav-Gal4>UAS-hTauV337M; ∆Pfdn515/40, and (H-H') Elav-Gal4>UAS-hTauV337M; UAS-α-Tubulin, ∆Pfdn515/40 double immunolabeled with neuronal membrane marker, HRP (green), and T46 antibody against total human Tau (magenta). The scale bar in H' for (F-H') represents 10 µm. Arrows point to the Tau aggregates. (I) Histogram showing the quantification of the number of Tau punctae per 100 µm2 normalized with HRP positive area in Elav-Gal4/UAS-hTauV337M (0.18±0.05), Elav-Gal4/UAS-hTauV337M; ΔPfdn515/40 (3.32±0.65), and Elav-Gal4>UAS-hTauV337M; UAS-α-Tubulin, ∆Pfdn515/40 (2.9±0.57). ***p<0.001; **p<0.01. At least 12 axons from three animals of each genotype were used for quantification.
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Figure 8—source data 1
Source data related to Figure 8.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-data1-v1.xlsx
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Figure 8—source data 2
PDF files containing original Western blots for Figure 8A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-data2-v1.zip
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Figure 8—source data 3
Original files for Western blot analysis displayed in Figure 8A.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-data3-v1.zip
Figure 8—figure supplement 1
Pfdn5 is required to stabilize microtubules at the synapses.
(A) Western blot showing protein levels of ace-Tubulin, α-Tubulin, and β-Tubulin in control, ∆Pfdn15/40, Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 and Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40. Ran protein levels were used as an internal loading control. (B) Histogram showing the percentage of α-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/40 (0.23±0.04), Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (0.75±0.16), and Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (1.05±0.04). ***p<0.001; *p<0.05. Three independent Western blots were used for quantification. (C) Histogram showing the percentage of β-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/40 (0.27±0.08), Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (1.03±0.23), and Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (1.01±0.01). ***p<0.001; *p<0.05. Three independent Western blots were used for quantification. (D) Histogram showing the percentage of ace-Tubulin normalized with Ran in control (1.00±0.00), ΔPfdn515/40 (0.34±0.13), Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (1.38±0.15), and Elav-Gal4>UAS-Pfdn5; ∆Pfdn515/40 (1.61±0.4). **p<0.01; *p<0.05. Three independent Western blots were used for quantification. (E-H') Confocal images of neuromuscular junction (NMJ) synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (E-E') control, (F-F') ΔPfdn515/40, (G-G') Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (H-H') Elav-Gal4>UAS-Pfdn5; ΔPfdn515/40 double immunolabeled for HRP (green), and CSP (magenta). The scale bar in H for (E-H') represents 10 µm. (I) Histogram showing number of satellite boutons from muscle 4 at A2 hemi segment in control (1.88±0.44), ΔPfdn515/40 (15.5±1.48), Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40 (14.25±1.39), Elav-Gal4>UAS-Pfdn5; ΔPfdn515/40 (3.53±0.32). ***p<0.001; ns, not significant. At least eight NMJs of each genotype were used for quantification. (J-J') Confocal images of third instar larval axons showing colocalization between Pfdn5 (magenta) and Tubulin-associated unit (Tau) (green). Scale bar in J' represents 10 µm. (K) Pearson’s correlation coefficient to quantify colocalization between Pfdn5 and axonal microtubule.
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Figure 8—figure supplement 1—source data 1
Source data related to Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-figsupp1-data1-v1.xlsx
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Figure 8—figure supplement 1—source data 2
PDF files containing original Western blots for Figure 8—figure supplement 1A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-figsupp1-data2-v1.zip
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Figure 8—figure supplement 1—source data 3
Original files for Western blot analysis displayed in Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104691/elife-104691-fig8-figsupp1-data3-v1.zip
Figure 9
A model depicting novel functional requirements of Pfdn5 in microtubule stabilization and its role in suppressing age-dependent neuropathy.
In axons, Pfdn5 physically associates with microtubules and stabilizes them, thereby suppressing the turnover of microtubules. The pathological Tau dislodges from microtubules in an age-dependent manner and forms pathological aggregates that induce neuronal death (Middle panel). Loss of Pfdn5 disrupts neuronal microtubules, resulting in abnormal synaptic morphogenesis and facilitating the dislodging of microtubule-associated Tau, resulting in the formation of Tubulin-associated unit (Tau) aggregates and stepping up the early onset of Tauopathies. An age-dependent reduction in the Pfdn5 levels or mutations in Pfdn5 could result in microtubule fragmentation and may facilitate Tau-induced neurotoxicity (Left panel). Pfdn5 suppresses Tau aggregation in a manner that involves microtubule stability and does not appear to regulate Tau solubility directly. Notably, neuronal overexpression of Pfdn5 suppresses the microtubule disruption even in aged flies, thereby inhibiting the progression of Tauopathy (Right panel).
Author response image 1
A double mutant combination of dTau and Pfdn5 aggravates the synaptic defects at the Drosophila NMJ.
(A-D') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (A-A') control, (B-B') ΔPfdn515/40, (C-C') dTauKO/dTauKO (Drosophila Tau mutant), (D-D') dTauKO/dTauKO; ∆Pfdn515/40 double immunolabeled for HRP (green), and CSP (magenta). The scale bar in D for (A-D') represents 10 µm.
Additional files
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Supplementary file 1
The table shows the list of HSPs used to screen as genetic modifiers of Tauopathies.
- https://cdn.elifesciences.org/articles/104691/elife-104691-supp1-v1.docx
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Supplementary file 2
Table shows the list of primers used in this study.
- https://cdn.elifesciences.org/articles/104691/elife-104691-supp2-v1.docx
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Supplementary file 3
Table shows details of RNAi lines used against cytoskeletal chaperones.
- https://cdn.elifesciences.org/articles/104691/elife-104691-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104691/elife-104691-mdarchecklist1-v1.pdf
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Prefoldin 5 is a microtubule-associated protein that suppresses Tau aggregation and neurotoxicity
eLife 13:RP104691.
https://doi.org/10.7554/eLife.104691.3
