Prefoldin cochaperones are genetic modifiers of Tau-induced neurodegeneration

(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.

(M) 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.

Loss of Pfdn5 disrupts microtubule organisation

(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 the 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 Fustch 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; ΔPfdn515/40 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.

(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/+; ΔPfdn515/40 (27.39 ± 2.21). ***p<0.001. At least 16 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, ΔPfdn15/40 and actin5C-Gal4>UAS-Pfdn5; ΔPfdn515/40. 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), ΔPfdn40/40(0.49 ± 0.04), ΔPfdn515/40 (0.46 ± 0.04), actin5C-Gal4/UAS-Pfdn5; ΔPfdn515/40 (1.05 ± 0.13). **p<0.01. Three independent Western blots were used for quantification.

(I) Histogram showing percentage of α-Tubulin normalised with Ran in control (1.00 ± 0.00), ΔPfdn515/15 (0.27 ± 0.04), ΔPfdn40/40 (0.16 ± 0.02), ΔPfdn515/40 (0.11 ± 0.04), actin5C-Gal4/UAS-Pfdn5; ΔPfdn515/40 (0.85 ± 0.07). ***p<0.001. Three independent Western blots were used for quantification.

(J) Histogram showing percentage of β-Tubulin normalised with Ran in control (1.00 ± 0.00), ΔPfdn515/15 (0.52 ± 0.04), ΔPfdn40/40 (0.39 ± 0.04), ΔPfdn515/40 (0.30 ± 0.09), actin5C-Gal4/UAS-Pfdn5; ΔPfdn515/40 (1.03 ± 0.11). **p<0.01. Three independent Western blots were used for quantification.

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; ΔPfdn515/40) 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.

Loss of Pfdn5 mimics and enhances Tau-induced synaptic defects

(A-H’) Confocal images of 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; ΔPfdn515/40, (D-D’) mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40, (E-E’) Elav-Gal4/+ (Gal4 control), (F-F’) Elav-Gal4>UAS-hTauV337M, (G-G’) Elav-Gal4>hTauV337M; ΔPfdn515/40, (H-H’) Elav-Gal4>hTauV337M;UAS-Pfdn5; ΔPfdn515/40 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; ΔPfdn515/40 (31.94 ± 1.18), mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40 (38.40 ± 2.17), Elav-Gal4/+ (28.13 ± 1.51), Elav-Gal4>UAS-hTauV337M (36.00 ± 2.65), Elav-Gal4>hTauV337M; ΔPfdn515/40 (60.34 ± 3.76), Elav-Gal4>hTauV337M; UAS-Pfdn5; ΔPfdn515/40 (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; ΔPfdn515/40 (2.94 ± 0.67), mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40 (15.6 ± 0.86), Elav-Gal4/+ (2.2 ± 0.38), Elav-Gal4>UAS-hTauV337M (14.06 ± 1.00), Elav-Gal4>hTauV337M; ΔPfdn515/40 (32.25 ± 3.2), Elav-Gal4>hTauV337M;UAS-Pfdn5; ΔPfdn515/40 (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; ΔPfdn515/40 (5.6 ± 0.2), mef2-Gal4>UAS-Pfdn5; ΔPfdn515/40 (5.8 ± 0.2), Elav-Gal4/+ (5.2 ± 0.4), Elav-Gal4>UAS-hTauV337M (6.4 ± 0.4), Elav-Gal4>hTauV337M; ΔPfdn515/40 (2.3 ± 0.2), Elav-Gal4>hTauV337M;UAS-Pfdn5; ΔPfdn515/40 (6.3 ± 0.3). ***p<0.001; ns, not significant. At least 12 NMJ of each genotype were used for quantification.

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; ΔPfdn515/40, (E-E’) Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5; ΔPfdn515/40 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 E and E’ point to the hTau punctae/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 6 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; ΔPfdn515/40 (J-J’’) Elav-Gal4>UAS-hTauV337M; UAS-Pfdn5; ΔPfdn515/40 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 8 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 8 animals of each genotype were used for quantification.

Over-expression of Pfdn5 or Pfdn6 suppresses age-dependent progression of hTau-induced neurodegeneration

(A-D) Bright-field images of 7 days old 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 7-day old 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-G) Bright field images of 14-day old Drosophila eyes expressing (E) GMR-Gal4>UAS- hTauV337M, (F) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (G) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6.

(E’-G’) Scanning electron microscopic images of 14-day old Drosophila eyes expressing (E’) GMR-Gal4>UAS-hTauV337M, (F’) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (G’) GMR- Gal4>UAS-hTauV337M; UAS-Pfdn6.

(H-J) Bright field images of 30-day old Drosophila eyes expressing (H) GMR-Gal4>UAS- hTauV337M, (I) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (J) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6.

(H’-J’) Scanning electron microscopic images of 30-day old Drosophila eyes expressing (H’) GMR-Gal4>UAS-hTauV337M, (I’) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn5, (J’) GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. The scale bar in J’ for (A-J’) represents 100 µm.

(K-L) Graph showing quantifications of age-dependent progression of ommatidial fusion (K) and percentage of degenerated eye area (L) in GMR-Gal4>UAS-hTauV337M, GMR-Gal4>UAS- hTauV337M; UAS-Pfdn5 and GMR-Gal4>UAS-hTauV337M; UAS-Pfdn6. Note that expression of Pfdn5 or Pfdn6 suppresses the Tau-induced neuronal death. At least 12 SEM eye images of each genotype were used for quantification at each time point.

(M) Histogram showing the percentage of fused ommatidia in 30 days old flies 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.

(N) Histogram showing percentage of degenerated area in 30 days old flies 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.

(O-R) Confocal images of a single section of 21-day old adult brain in (O) Elav-Gal4/+ (control), (P) Elav-Gal4>UAS-hTauV337M, (Q) Elav-Gal4>hTauV337M; UAS-Pfdn5, (R) Elav-Gal4>hTauV337M; UAS-Pfdn6 double immunolabeled with Hoechst (cyan), and Phalloidin (magenta). The insets represent the 3X magnified portion of the image. Arrows in the figure P inset point to the pathological vacuolar structures. The scale bar in R for (O-R) represents 10 µm.

(S) Histogram showing the quantification of number of vacuoles in 21 days old adult brain in GMR-Gal4/+ (4.67 ± 1.45), UAS-hTauV337M/+; GMR-Gal4/+ (35.67 ± 5.04), UAS- hTauV337M/+; GMR-Gal4/UAS-Pfdn5 (3.75 ± 0.48), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 (5.00 ± 1.47). ***p<0.001. At least three brains of each genotype were used for quantification.

(T) Histogram showing the quantification of vacuole size (in µm2) in 21-day old adult brain in GMR-Gal4/+ (9.27 ± 1.41), UAS-hTauV337M/+; GMR-Gal4/+ (328.7 ± 82.24), UAS- hTauV337M/+; GMR-Gal4/UAS-Pfdn5 (19.4 ± 4.2), UAS-hTauV337M/+; GMR-Gal4/UAS-Pfdn6 (16.92 ± 0.89). **p<0.01. At least three brains of each genotype were used for quantification. The raw images associated with Figure 6A-6J’ is represented in Supplemental 1-4 zip files.

Over-expression of Pfdn5 or Pfdn6 rescues Tau-induced defects in learning and memory

(A) Cartoon of 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 [57]. During training, flies are exposed to a normally attractive odorant 2,3-butanedione (10-3-fold dilution) in a spaced training protocol: 8X repeats of a training trial involving 5 minutes in the presence 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 over-expression 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.

Microtubule stability and 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, ΔPfdn15/40, and Elav-Gal4>UAS-α-Tubulin; ΔPfdn515/40. Ran protein levels were used as an internal loading control.

(B-D’) Confocal images of 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 shows 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 6 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; ΔPfdn515/40, and (H-H’) Elav-Gal4>UAS-hTauV337M; UAS-α- Tubulin, ΔPfdn515/40 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, ΔPfdn515/40 (2.9 ± 0.57). ***p<0.001; **p<0.01. At least 12 axons from 3 animals of each genotype were used for quantification.

A model depicting novel functional requirement 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 dislodging of microtubule-associated Tau, resulting in Tau-aggregates formation 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).