Autophagy is ineffective for clearance of nuclear aggregates. (A) Atg8/LC3 colocalizes with cytoplasmic but not nuclear Atx-1(Q85). HeLa cells expressing FLAG-Atx1(Q85) (Top and Middle) or Atx1(Q85) containing a mutant nuclear localization sequence (K772T) (Bottom) were imaged for anti-FLAG immunofluorescence (Left) and Atg8/LC3 immunofluorescence (Center). Nuclei were labeled with bisbenzimide staining (Right). Nuclear (arrowheads) and cytoplasmic (arrows) inclusions are indicated. (Scale bar, 1 μm.) (B) Atg knockdown increases aggregation of cytoplasmic but not nuclear Atx1(Q85). Shown are filter retardation assay of HEK-293 cells transiently expressing the indicated Atx1 constructs and treated with the indicated siRNA. *, P < 0.0001; **, P = 0.0003 vs. Atx1(Q85).

Cytoplasmic but not nuclear inclusions from diseased brains recruit Atg8/LC3. (A) Cortex from 16-week-old R6/2 mouse was stained with EM48 anti-Htt antibody (red, Left) and Atg8/LC3 antibodies (green, Center). A representative image of cortical neurons is shown. (B) Cortex from 1-year-old Hdh^(CAG)150 knock-in mouse stained with anti-ubiquitin (Ub, red) and Atg8/LC3 (green). Nuclei are stained with bisbenzimide. (C) Cerebellum from Atx1-overexpressing mouse was stained with anti-ubiquitin (Ub, red) and Atg8/LC3 (green). A representative image of Purkinje cells is shown. (D) A midbrain section from a Parkinson's disease patient was stained with LB509 anti-α-synuclein and Atg8/LC3 antibodies as indicated. The image is representative of two sections from four different patients. Arrows indicate cytoplasmic inclusions, and arrowheads indicate nuclear inclusions. (Scale bar, 2 μm.)

Atgs are recruited to Htt IBs. (A) IBs in Q103-expressing cells are surrounded by Atg8/LC3 staining material. Shown are micrographs of HeLa cells transiently transfected with GFP-Htt(Q25) (Upper) or GFP-Htt(Q103) (Lower, green) and stained with antibodies to Atg8/LC3 (red) or bisbenzimide to label nuclei (blue). (Scale bar, 10 μm.) (B) Htt IBs are immunopositive for other Atg proteins. Shown are micrographs of HeLa cells transiently transfected with GFP-Htt-Q103 (green) and stained with antibodies to the indicated Atgs (red) or bisbenzimide to label nuclei (blue). (Scale bar, 10 μm.) (C) Analysis of Atg8/LC3 localization to a GFP-Htt(Q103) IB by digital deconvolution microscopy. (Right) A projection from a reconstruction of optical sections. (Scale bar, 1 μm.) (D) Colocalization of Atg8/LC3 (Center, red) in HeLa cells transiently transfected with myc-tagged Atx3(Q84) (Top) or HEK-293 cells stably expressing GFP-CFTRΔF508 (Middle) or TCRα-GFP (Bottom). CFTR and TCRα cells were treated overnight with the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN; 10 μg/ml) to enhance aggregation. (Scale bar, 2 μm.)

Autophagy is essential for aggregate clearance. (A) Atg8/LC3 knockdown increases aggregate burden. Effect of Atg8/LC3 siRNA on GFP-Htt(Q71) aggregation assessed by filter retardation is shown. HEK-293 cells stably expressing GFP-Htt(Q71) were treated with the indicated amount of siRNA and evaluated for Atg8/LC3 or GFP-Htt(Q71) expression by Western blot (WB) or filter-trapped GFP-Htt(Q71) (FT). Intensities (relative to 0 siRNA) of filter-retained spots were quantified by image analysis (Upper, corresponding spots) and plotted (Lower). *, P < 0.0001 vs. control. n = 3. (B) Effect of Atg8/LC3 siRNA on GFP-Htt(Q71) aggregation assessed by IB frequency. Cells with IBs (representative field, GFP, Top) were counted (n = 300 from three independent trials) and quantified (Bottom). Bisbenzimide staining (nuc, Middle) shows the total number of nuclei in representative microscopic fields. *, P = 0.0049. (Scale bar, 50 μm.) (C) Atg5 knockdown increases aggregated burden. Effect of HAtg5 siRNA on GFP-Htt(Q71) aggregation assessed by filter retardation is shown. HEK-293 cells stably expressing GFP-Htt(Q71) were treated with 0.8 μg of siRNA and evaluated for HAtg5/12 conjugate expression (Top), or filter-trapped GFP-Htt(Q71) (Middle). Intensities (relative to lacZ siRNA) of filter-retained spots were quantified by image analysis (of the corresponding spots) and plotted (Bottom). *, P < 0.0001. n = 3. (D) Effect of Atg knockdown on IB frequency in differentiated neuro2a cells induced for 4 days to express GFP-Htt(Q150). Cells were transfected with the indicated siRNA on 2 consecutive days as indicated by the timeline in F and counted (n = 100 per condition) for the presence of IBs. *, P = 0.0064; **, P = 0.0032 vs. control. (E) Effect of siRNA on GFP-Htt(Q150) aggregation assessed by filter retardation. Filter retardation assay was performed on lysates of neuro2a cells treated with the indicated siRNAs 5 days after removal of inducer. *, P < 0.0001 vs. control. (F) Atg knockdown impairs clearance of preformed aggregates. Differentiated neuro2a cells were induced to express GFP-Htt(Q150) for 3 days (ON, timeline at bottom), followed by 6 days in culture without inducer (OFF). Cells were transfected with indicated siRNA on days 0, 1, 4, and 7 (upward arrows) and were scored for the presence of IBs by microscopy from days 3–9. Data are normalized to 100% on day 4 for each condition to permit comparison of rates. *, P = 0.0006; **, P = 0.0007 vs. control. Absolute levels on day 4 are shown in D.(Inset) Effect of siRNA on steady-state HAtg5/12 (Upper) and Atg8/LC3 (Lower) levels. Cell lysates from cells transfected with Atg (+) or lacZ (–) siRNA on day 5 were analyzed by immunoblotting using antibodies to Atg12 or Atg8/LC3 or actin for loading control.