Journal: eLife
Article Title: Prefoldin 5 is a microtubule-associated protein that suppresses Tau aggregation and neurotoxicity
doi: 10.7554/eLife.104691
Figure Lengend Snippet: ( 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 . 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 CuSO 4 +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-hTau V337M /+ ; 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-hTau V337M ; 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 hTau V337M expression rescues the learning and memory deficits in ( F ) Elav-Gal4/UAS-hTau V337M ; UAS-Pfdn5/+ ; naïve flies (14.28±1.96), trained flies (–1.5±1.7). Consistently, pan-neuronal over-expression of Pfdn6 along with hTau V337M expression also rescues the learning and memory deficits in ( G ) Elav-Gal4/UAS-hTau V337M ; 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-hTau V337M Grade I (Grade I represents the defective mushroom body with one α-lobe missing), ( K-K' ) Elav-Gal4>UAS-hTau V337M Grade II (Grade II represents the severe defects in mushroom body with both α-lobe missing), ( L-L' ) Elav-Gal4>UAS-hTau V337M ; UAS-Pfdn5 , ( M-M' ) Elav-Gal4>UAS-hTau V337M ; 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-hTau V337M (91.67±8.33), Elav-Gal4>UAS-hTau V337M ; UAS-GFP (93.75±6.25), Elav-Gal4>UAS-hTau V337M ; UAS-Pfdn5 (0.00±0.00), Elav-Gal4>UAS-hTau V337M ; 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-hTau V337M (NBs: 11.1, Grade I: 22.2%, Grade II: 66.7), Elav-Gal4>UAS-hTau V337M ; UAS-Pfdn5 (NBs: 100, Grade I: 0, Grade II: 0), Elav-Gal4>UAS-hTau V337M ; UAS-Pfdn6 (NBs: 83.3, Grade I: 16.6, Grade II: 0). Figure 7—source data 1. Source data related to .
Article Snippet: Other primary antibodies used in this study are mouse anti-dPfdn5 (this study, 1:200), mouse anti-ace-tubulin (1:500, Sigma-Aldrich, Missouri, USA), anti-Tau (T46, 1:100, Invitrogen, Waltham, MA, USA), anti-FasII (1:50, DSHB), anti-D5D8N (1:500, CST, Boston, MA), and anti-phospho-Tau (AT8, 1:100, Invitrogen, Waltham, MA, USA).
Techniques: Control, Expressing, Variant Assay, Over Expression, Labeling, Immunolabeling