(a) Immunoblot for the indicated proteins in fractions enriched in autophagosomes (APH) and autophagolysosomes (APHL) isolated from liver of ¹⁸Qhtt and ¹¹¹Qhtt mice. Bottom: Changes (folds increase or decrease) in the levels of each protein. Mean+s.d. n = 4. (b) Homogenates (Homo), cytosol (Cyt) and fractions enriched in autophagosomes (APH) and in autophagolysosomes (APHL) isolated from wild type (¹⁸Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt) livers were subjected to immunoblot for htt. A representative immunoblot of 4 experiments with duplicated samples is shown. * p < 0.05. Full–length blots are presented in Supplementary Fig. 20.

(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of ¹⁸Qhtt and ¹¹¹Qhtt mice. Insets: higher magnification images of single vesicles. Right: Percentage of vesicles with an electron–clear (light), vesicular (multivesicular) or electron–dense content (dark). Mean+s.e.m of 3 different isolations (estimated > 1,000 AVs). (b) Bidimensional electrophoresis and SyproRuby staining of the same fractions. Samples on the right are the content of the same fractions isolated in the supernatant after hypotonic shock and high speed centrifugation. * p < 0.05.

(a–d) Degradation of long–lived proteins in MEFs from wild type (¹⁸Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt). (a–b) Rates of protein degradation after serum removal (a) or treatment with rapamycin or thapsigargin (b). (c) Lysosomal degradation calculated as degradation sensitive to NH₄Cl. (d) Contribution of macroautophagy calculated when adding 3–methyladenine (3–MA). (e) Degradation of long–lived proteins in striatal cells from wild type (⁷Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt) in response to different autophagic stimuli. (f–g) Rates of protein degradation in wild type and HD MEFs (f) and striatal cells (g) control or after RNAi for Atg7 (Atg7 (–)). (h) Contribution of macroautophagy to the degradation of long–lived proteins in lymphoblasts from normal control (NC) and from HD patients. All values are expressed in percentage and are mean+s.d. of values from 2–4 different individuals and 3–4 different experiments. *,^# significant with untreated (*) or control (#) for p < 0.05.

(a) Electron micrographs of striatal neurons from control and HD94 mice grown over a wild type rat astrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Right: number of vacuoles per cell profile (13–17 cell profiles in triplicate experiments). (b) Electron micrographs of striatal cells from wild type (⁷Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt). Right: Higher magnification fields. (c) Electron micrographs of lymphoblasts from normal control (NC) and HD patients maintained in the presence or absence of serum. Right: higher magnifications areas containing enlarged electron–clear vesicles (arrows). (d) Immunogold for LC3 in striatal cells from mutant huntingtin knock–in mice (¹¹¹Qhtt), striatal neurons from HD194 mice and lymphoblasts from HD patients. Full fields and more details of autophagic vacuoles are shown in Supplementary Fig. 10 and 11. * p < 0.05.

(a, d) Immunoblot for htt or p62 of total autophagosomes (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from wild type (¹⁸Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt) livers. Left: Representative immunoblots. Right: Distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six different immunoblots as the ones shown here. Values are mean+s.d. * p<0.05 compared to wild type values. (b) Filter retardation analysis of the same fractions as in a, and blotted for htt. Negative signal indicated absence of aggregates retained in the filter. (c) Immunoblot for ubiquitin in homogenates (homog), cytosol and the autophagic fractions described in a, was performed in a 6% (top) and a 16% (bottom) gel. Dotted line indicates separation between the stacking and running part of the gel. (d) Immunoblot for LC3 and p62 of homogenates (Homo) and autophagosomes (APH) isolated from wild type (¹⁸Qhtt) and mutant huntingtin knock–in mice (¹¹¹Qhtt) brains. (e) Membranes of autophagic vacuoles shown in d were subjected to immunoprecipitation for htt in mild co–immunoprecipitation buffer. Note that input for ¹¹¹Qhtt AVs was 1/3 of the input used for ¹⁸Qhtt to avoid problems with antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full–length blots are presented in Supplementary Fig. 21.

(a) LC3 immunostaining of ¹⁸Qhtt and¹¹¹Qhtt MEFs maintained in the presence (+) or absence (−) of serum and lysosomal proteolysis inhibitors. Bottom: mean number per cell (left) and average size (right) of LC3 positive vesicles. n = 4 (b) LC3 immunoblot in the same cells after serum removal or thapsigargin (TG) treatment. PI: protease inhibitors. Bottom: LC3–II levels and LC3–II flux (dotted lines). N = 4. (c) LC3–II values and LC3–II flux (dotted lines) in neuronal cultures from wild type (WT) or HD94 mice (HD) grown over wild type rat astrocyte monolayers analyzed as in b. n = 4. (d) LC3 staining of lymphoblasts from 3 normal control (NC) or HD patients maintained in the presence or absence of serum. Right: mean number of LC3 positive vesicles. Extended study in Supplementary Fig. 8b,c. (e) LC3–II levels in NC and HD lymphoblasts treated or not with protease inhibitors. Right: LC3–II values and LC3–II flux (dotted lines). Extended study in supplementary Fig. 8a. Values are all expressed as mean+s.d. Differences with control, where significant, are indicated with ^# for p < 0.05. Full–length blots are presented in Supplementary Fig. 20.

(a) COX IV immunofluorescence in ¹⁸Qhtt and ¹¹¹Qhtt MEFs maintained in the presence or absence of serum. Bottom: Number of mitochondria per cell. Mean+s.d. of 10–20 cells in 3 different experiments. (b) Electron micrographs of striatal neurons from wild type and HD94 mice grown over a wild type rat astrocyte monolayer. Arrows: Abnormally short (black) or abnormally long (green) mitochondria. Right: Number of mitochondria per cell profile. Mean+s.d. of 10 cells per group in triplicates. (c) Left: Electron micrographs of lymphoblasts from normal control or HD patients. Arrows are as in b. Right: Percentage of cellular area occupied by mitochondria in different individuals. Line indicates the mean value of the population. (d, e) Striatal cells from ⁷Qhtt and ¹¹¹Qhtt mice (d) and MEFs from ¹⁸Qhtt and ¹¹¹Qhtt mice (e) co–stained with mitotracker and mito–ROS. Right: Merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control of depolarization. Differences with control are significant for * p < 0.05. (f,g) Striatal cells from ⁷Qhtt and ¹¹¹Qhtt mice untreated or treated with vinblastine were co–stained for LC3 and mitotracker (f) or Bodipy 493/503 (g). Arrows point to colocalization events. Extended study in Supplementary Fig. 16.

(a,c–e) Neutral lipids in MEFs from ¹⁸Qhtt and ¹¹¹Qhtt mice (a), striatal cells from ⁷Qhtt and ¹¹¹Qhtt mice (c), primary striatal neurons from ¹⁸Qhtt and ¹¹¹Qhtt mice grown in a monolayer of their own astrocytes (d) and lymphoblasts from a control and HD patient (e) were stained with Bodipy 493/503. MAP2 staining highlights neurons. Extend study is shown in Supplementary Fig. 15b. (b, f) Electron micrographs of livers from ¹⁸Qhtt and ¹¹¹Qhtt mice (b) and lymphoblast from a HD patient (with 78/15 polyQ repeats) (f). LD: lipid droplets (green arrows). Right (in b): Number of LD, mean area of LDs and percentage of cellular area occupied by LD. Mean+s.d. n = 3. (g) Fraction of cellular cytosol occupied by LD quantified in 3 NC and 4 HD. Numbers of polyQ repeats are shown at the bottom. Mean+s.d. of >100 cell profiles. Dotted red lines: mean value of all NC and HD patients. * p < 0.05. (h) Oil Red O staining of striatal tissue from brain of normal control (top) and two HD patients (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are indicated in the right panels with green arrows. (i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell was calculated for each of the samples by quantification of 8–9 different fields. *p < 0.001, ANOVA, p < 0.01 for each HD patient vs. each control patient, neither control nor HD patients were different from each other, Tukey post–hoc test.