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First published online 22 April 2008
doi: 10.1242/jcs.025726

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Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease

¹ Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0XY, UK
² Department of Genetics, University of Cambridge, CB2 3EH, UK

View larger version (23K): [in this window] [in a new window]   Fig. 1. Rab5 modulates the aggregation and toxicity of mutant huntingtin. (a) Quantification of GFP-expressing COS-7cells showing signs of cell death that had been transiently transfected with dominant-negative (DN), constitutive active (CA) or wild-type (WT) Rab5, or empty vector control, and the EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours. ***P<0.0001, **P<0.001, *P<0.05. (b) Quantification of GFP-expressing COS-7 cells containing aggregates that were quantified for toxicity shown above. ***P<0.0001. (c) Quantification of GFP-expressing HeLa cells containing aggregates transiently transfected with siRNA targeting Rab5a, Rab5b, Rab5c or all three siRNA simultaneously (Rab5abc) for 72 hours, and also with EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats for the last 24 hours of the 72-hour siRNA transfection period. ***P<0.0001, *P<0.05. (d) Rab5 overexpression increases the numbers of rhabdomeres in ommatidia of mutant huntingtin-expressing flies. Frequency distribution of ommatidia with different numbers of rhabdomeres three days after eclosion (hatching) in progeny of flies that express mutant huntingtin exon 1 (gmrQ120) and that had been crossed to either a control stock (w1118) (white minus; have huntingtin transgene only) or to Rab5-EGFP flies (have huntingtin and Rab5 transgenes). P0.001, t-test; P0.001, Mann-Whitney U test. The rhabdomere frequency of Rab5 flies crossed to a control stock is also shown. (e) Odds ratio of GFP-expressing cells with Q74 aggregates in wild-type (Atg5+/+) vs Atg5 knockout (Atg5–/–) MEFs. ***P<0.0001. (f) Odds ratio of Q74-expressing Atg5–/– (Atg5 knockout) or Atg5+/+ (wild-type) MEF cells with aggregates, after transient transfection with dominant-negative Rab5 (DN-Rab5), constitutive active Rab5 (CA-Rab5) or empty vector control and EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48 hours. ***P<0.0001. Odds ratios are given to compare pooled summary statistics across multiple independent experiments (see Materials and Methods). Control conditions are fixed at 1 in both cell lines to facilitate comparisons. (g) Quantification of GFP-expressing HeLa cells containing aggregates that had been transiently transfected with dominant-negative Rab5 (DN-Rab5) or empty vector (Cont) and huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours and were either left untreated (–Rap) or treated with 0.2 µg/ml rapamycin (Rap) to induce autophagy. **P<0.001. Error bars in all graphs represent the s.e.m.

View larger version (171K): [in this window] [in a new window]   Fig. 2. (a) COS-7 cells were transiently transfected with empty vector (Control), DN-Rab5, CA-Rab5 or WT-Rab5 and GFP-LC3 or mRFP-LC3 (3:1 ratio) for 24 hours. GFP-positive or mRFP-positive cells with increased numbers of LC3-positive vesicles (>20 vesicles per cell) were counted. 29% of control cells had >20 vesicles per cell. ***P<0.0001. (b) Western blot analysis of COS-7 cells co-transfected with empty vector (C), DN-Rab5, CA-Rab5 or WT-Rab5 and Myc-LC3 for 24 hours in the presence of 200 nM bafilomycin A1 (treated for last 15 hours), using anti-Myc antibody. GFP was used as a transfection control. Representative image from three independent experiments; quantification of the band intensities from these experiments represented as LC3-II:GFP ratio is shown in the graph; *P<0.05. (c) Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector [either untreated or treated for 24 hours with 10 mM 3-methyladenine (3-MA)] or with dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. An increased abundance of large punctate Atg5 structures can be noticed with DN-Rab5 and 3-MA treatment. (d) HeLa cells were transfected with siRNA targeting Vps34 or control siRNA for 48 hours after which GFP-Atg5 together with siRNA was transfected for further 24 hours. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of Q74-expressing HeLa cells with aggregates is shown in the graph. Cells were transiently transfected with control siRNA or siRNA targeting Vps34 for 48 hours and with HA-tagged huntingtin exon 1 containing 74 polyglutamine repeats for further 24 hours. ***P<0.0001. (e) Colocalisation of GFP-Atg5 structures (green) with Myc-tagged FYVE (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Myc-FYVE for 24 hours. (f) Colocalisation of GFP-Atg5 structures (green) with beclin 1 (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Flag-tagged wild-type (WT) beclin 1. (g) Colocalisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15 hours. In panels e-g we observed >30% colocalisation between GFP-Atg5 structures and saponin-extracted, membrane-associated, FYVE, beclin 1 or Rab5 in cells that expressed both of the respective proteins. (h) Colocalisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells co-transfected with DN-Rab5 and GFP-Atg5 and HA-tagged Atg12 for 24 hours. Nuclei labelled with DAPI are in blue.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. (a) HeLa cells transfected with control or Atg7 siRNA for 48 hours were subjected to a second round of transfection with GFP-Atg5 together with the siRNA for another 24 hours. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). (b) HeLa cells transfected with control vector or DN-Rab5 together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The ratio of Atg5-Atg12 versus free Atg12 is shown in the graph. Data are from four independent experiments; ***P<0.0001. (c) Atg5-Atg12:Atg12 ratio obtained from experiments similar to those shown in b, but performed in the presence or absence (control) of 3-methyladenine (3-MA; four independent experiments; left), or with control siRNA or siRNA targeting Vps34 (three independent experiments; right); ***P<0.0001, **P<0.001. (d) HeLa cells transfected simultaneously with siRNA targeting Rab5a, Rab5b and Rab5c (Rab5; left), Atg7 (right) or control siRNA for 48 hours were subjected to a second round of transfection with Atg5 and HA-Atg12 together with the Rab5 or Agt7 siRNA for another 24 hours. Western blot analysis was performed using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. (e) Immunostaining of HeLa cells transfected with either control (in the prescence or absence of 3-MA) or DN-Rab5 and HA-Atg12 for 24 hours after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. (f) Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24 hours after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Arrows indicate punctate Atg12 structures in control cells. Quantification of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 in percent are given in the bar graph. ***P<0.0001.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Rab5 is part of a macromolecular complex containing Vps34 and beclin 1. (a) Colocalisation of endogenous Rab5 (green) and endogenous beclin 1 (red) in 3MA-treated HeLa cells after saponin extraction. DAPI-stained nuclei are in blue. (b) Colocalisation of constitutive active Rab5 (CA-Rab5; green) with wild-type beclin 1 (WT-Beclin, red). (c) COS-7 cells transfected with control vector alone (lane 1) or Flag-tagged wild-type beclin 1 alone (lane 2) or wild-type beclin 1 with wild-type Vps34 (lane 3) were immunoprecipitated using anti-Flag antibody (to immunoprecipitate beclin 1) and blotted for Rab5 using an anti-Rab5 antibody. (d) Q74-HA aggregation in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 as described in Fig. 2d; P<0.005. (e) Ratio of Atg5-Atg12 conjugate to Atg12 (data from three independent experiments) in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 for 72 hours. Atg5 and HA-Atg12 were transfected for the last 24 hours. P=0.0003.

View larger version (37K): [in this window] [in a new window]   Fig. 5. (a) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative dynamin II (DN-Dyn-II) or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. (b) Distribution of GFP-LC3 vesicles in control (left) or DN-dynamin (red) transfected cells (right). (c) COS-7 cells were transiently transfected with empty vector (Control), DN-dynamin (DN-Dyn) and GFP-LC3 (3:1 ratio) for 24 hours. GFP-positive cells with an increased number of LC3-positive vesicles (>20 vesicles per cell) were quantified; ***P<0.0001. (d) Lysates from HeLa cells expressing empty vector control (Cont) or DN-dynamin (DN-Dyn) were blotted for endogenous LC3, and actin as control. Quantification of band intensities from four independent experiments is shown; **P<0.001. Under exposure conditions that allow endogenous LC3-II quantification in these cells, the LC3-I signal is frequently too low to be detected. (e) NRK cells were transiently transfected for 15 hours with either empty vector [control; with or without bafilomycin A1 (BafA1)] or DN-dynamin (DN-Dyn), and mRFP-LC3 and GFP-lgp120. mRFP-LC3 and GFP-lgp120 double-stained vesicles in individual cells are given in percent (Jahreiss et al., 2008). ***P<0.0001. (f) HeLa cells transfected with control vector or DN-dynamin (DN-Dyn) together with HA-Atg12 and Atg5 were subjected to western blot analysis using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (g) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative (DN) Vps4 or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001.

View larger version (23K): [in this window] [in a new window]   Fig. 6. (a) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 48 hours were subsequently transfected with EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (Q74) for 24 hours. Quantification of Q74 expressing cells with aggregates is shown in the graph. *P<0.01. (b) HeLa cells stably expressing GFP-LC3 were transfected with control or clathrin siRNA for 72 hours, numbers of LC3-positive vesicles were counted (>20 vesicles per cell); ***P<0.0001. (c) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 72 hours were subjected to western blot analysis using anti-LC3 and anti-actin antibodies. Under exposure conditions that allowed endogenous LC3-II quantification in these cells, the LC3-I signal was frequently too low to be detected. Quantification of band intensities from three independent experiments is shown. *P<0.05. (d) HeLa cells transfected with control siRNA (Cont) or siRNA targeting clathrin heavy chain (Cla) for 48 hours were subsequently transfected with HA-Atg12 and Atg5 for a further 24 hours. Western blot analysis was performed with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (e) HeLa cells stably expressing GFP-LC3 were left untreated (Cont) or treated with 5 mM methyl-β-cyclodextrin (β-CD), in the presence (+) or absence (–) of bafilomycin A1 (BafA1) for 6 hours were subjected to western blot analysis using anti-GFP (to detect LC3) and anti-actin antibodies. (f) Lysates from HeLa cells transfected with empty vector (Cont), DN-Rab5 or CA-Rab5 were blotted for phosphorylated (S6-P) and total (S6-T) ribosomal protein S6. Lysate from cells treated with rapamycin (Rap) was used as a positive control. (g) HeLa cells transfected with control vector (Cont) or wild-type Rheb together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (h) Schematic hypothetical representation of how autophagy is regulated by Rab5. Our data suggest that the Atg5 structures are probably precursors of the pre-autophagosomal structures. The accumulation of Atg5 structures that were observed by us following inhibition of Rab5 or Vps34 might be owing to a block in the progression from early Atg5-positive autophagosomal structures to the formation of autophagic vacuoles.

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