Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

Summary Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

. Analysis of senescence markers in primary culture mouse fibroblasts. (A-C) βgalactosidase staining in fibroblasts isolated from 4 m and 24 m old mice. (A) Representative images of phase, β-galactosidase and DAPI staining. Two independent mice for each age are shown. Senescent IMR-90 fibroblasts at population doubling level (PDL) 50 were used as control for positive staining. Bar: 10 µm. Quantification of percentage of β-galactosidase stained cells (B) and intensity of staining normalized to senescent IMR-90 control cells (C) (n=4). (D) Immunoblot for the indicated senescence markers proteins in the same fibroblasts. Bottom: Quantification of levels of each protein relative to those in 4 m old mice. All values are mean ± s.e.m. Two-tailed unpaired Student's t-test (for single comparisons) was applied. Differences were significant for **p<0.01 and ***p<0.001. Absence of symbols indicate no significative difference. presence/absence of serum for 4 hours and immunostained for LAMP1 (green). LAMP1 staining (top), merge with nuclei staining with DAPI (grey) (middle) and higher magnification images of the boxed area (bottom). Bar: 10 µm. (C) Primary fibroblasts from 4 m and 24 m old mice were transfected with the tandem reporter mCherry-GFP-LC3 and cultured in presence or absence of serum. Left: representative images of single channels. Dashed white lines indicate the perinuclear region and continuous white lines the cell profile. Right: Quantification of the percentage of the area of autophagosomes (APG; mCherry+ GFP+ vesicles) and autolysosomes (AUT; mCherry+ GFPvesicles) located in the perinuclear cellular region (IN). All values are mean ± s.e.m. Two-tailed unpaired Student's t-test (for single comparisons) or one-way analysis of variance and Bonferroni post hoc test (for multiple comparisons) were applied. Differences were significant for *p<0.05, **p<0.01 and ***p<0.001. Absence of symbols indicate no significative difference. Figure S3. Cytoskeleton, motor proteins and autophagic vacuole fusogenicity in primary fibroblasts from old mice. (A-C) Primary fibroblasts derived from 4 m and 24 m old mice immunostained for tubulin (A, B) or acetylated tubulin (C). DAPI stained nuclei are shown in grey in A and C Bar: 10 µm. The microtubular organizing centers (boxed areas in A) are shown at higher magnification in B. Bar: 2 µm. (D) Immunoblot for the indicated molecular motors in total cellular lysates from the same cells as in Fig. S1. Bottom: Quantification of levels of each protein relative to those in 4 m old mice. Values are all mean ± s.e.m. Two-tailed unpaired Student's t-test was used. Absence of symbols indicate no significative difference. Figure S4. Association of molecular motors to autophagic compartments. A-B. Immunoblot for dynein (Dyn) of autophagosomes (APG) and autolysosomes (AUT) isolated from livers of normally fed or 24h starved mice (A) and in membranes (Mb) and matrices (Mtx) of these compartments separated by a hypotonic shock and collected by centrifugation (B). Hom: homogenate. (C) APG derived from 4 m old mice immunostained for LC3 (green) and dynein (red). Single channels and merged are shown. Boxed area is shown at higher magnification to illustrate colocalization between both markers.   Fig. 6I. Bar: 10µm. All values are mean ± s.e.m.One-way analysis of variance and Bonferroni post hoc test (for multiple comparisons)was applied. Differences were significant for *p<0.05 and ***p<0.001. Absence of symbols indicate no significative difference.

Supplementary Videos
Supplementary Video 1. Motility of APG isolated from livers of 4 m old mice in an in vitro motility system. Liver vesicles were flowed into a 5 μl microscopy chamber pre-coated with Taxol-stabilized fluorescence microtubules (red), and after binding, these were stained with LC3 antibody (green).

Supplementary Video 2.
Motility of APG isolated from livers of 24 m old mice in an in vitro motility system. Liver vesicles were flowed into a 5 μl microscopy chamber pre-coated with Taxol-stabilized fluorescence microtubules (red), and after binding, these were stained with LC3 antibody (green).

Cell culture
Cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10 % Fetal bovine serum (FBS), 50 μg ml−1 penicillin, and 50 μg ml−1 streptomycin at 37 °C with 5 % CO2. All cells were tested for mycoplasma contamination using a DNA staining protocol with Hoechst 33258 dye. When indicated, cells were washed three times with Hank's balanced salt solution (HBSS; Invitrogen, Carlsbad, CA) and placed in fresh medium without serum to activate the autophagic process. Where indicated, lysosomal proteolysis was inhibited by addition of 20 mM NH4Cl and 100 µM leupeptin (Fisher Scientific). The β-galactosidase staining kit (Cell Signaling Technology) was used to measure senescence in primary cultures. Senescent IMR-90 cells (population doubling level 50) were used as positive control.
Formaldehyde and paraformaldehyde were from PerkinElmer Life and Analytical Sciences (Waltham, MA).

Cell transfection and RNA interference.
Cells were transfected with cDNA constructs mt-Keima (Katayama et al. 2011) and mCherry-GFP-LC3 (Kimura et al. 2007) using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Knock-down was performed using lentiviral-delivered small hairpin RNA (shRNA) from the Mission-Sigma library (Sigma-Aldrich) against KIFC3 (5′-CAACGACTACAATGGGCTCAA-3′) (TRCN0000116466) and transduction with virus carrying the empty vector was used as control. Lentiviral particles were generated by co-transfection with the third-generation packaging constructs pMDLg/pRRE and pRSV-REV, and as envelope the G glycoprotein of the VSV (pMD2.G) into HEK293T cells as described before (Massey et al. 2008).

Isolation of subcellular fractions from mouse liver
Autophagosomes (APG), autolysosomes (AUT) and lysosomes were isolated from liver of fed or 6-h-fasted mice by centrifugation in a discontinuous metrizamide density gradient as described previously (Marzella et al. 1982). The homogenate was then centrifuged 2500 x g for 10 min, and the post nuclear supernatant was collected, mixed with 80% Nycodenz (Accurate Chemical and Scientific Corp, Westbury, MA) and loaded into the bottom of a Nycodenz step gradient consisting of 50, 24.1, 17.2, and 0 % Nycodenz (w/v) in MEPS. This was centrifuged 200,000 x g for 2 hours. The cloudy layer at each interface was collected and frozen as 15 µl single use aliquots. Vesicles floating above 17.2 % Nycodenz were used for motility assays.

Vesicle Motility Assay
Motility assays were performed in a 3 μl optical chamber flow cells consisting of 24x50 mm coverglass on the bottom, two strips of double-sided tape to from a channel and cut glass to form a cover as described previously (Murray & Wolkoff 2007). Fluorescent microtubules in MT buffer (80 mM K2-PIPES, 1 mM EGTA, 1mM MgCl2, 3% glycerol, 1mM GTP, pH 7.0) were flowed into chambers coated with 30µg DEAE-dextran to allow their attachment. Chambers were washed and vesicles were flowed in and allowed to bind to the microtubules on ice for 15 min. Chambers were washed and vesicles were then labeled with primary antibody to LC3 and p62, by washed, and visualized by and fluorescent secondary antibody. For image acquisition, chambers were removed from ice and placed on the fluorescence microscope stage maintained at 37°C. Time-lapse image collection was initiated followed by addition of 50 µM ATP, to induce motor dependent vesicle motility. Motility assay buffer for washes consisted of PMEE buffer (35 mM K2 -PIPES, 5 mM MgCl2 , 1 mM EGTA, 0.5 mM EDTA, pH 7.4), plus 2 mg/mL bovine serum albumin, 20 µM Taxol, 4 mM DTT, 2 mg/mL Na-ascorbic acid. CoolSNAP HQ cooled CCD camera (Photometrics, Roper Scientific, Tucson AZ). The system was also run with Metamorph Software, an Olympus iX71 temperature enclosed stand heated to 37⁰C, and a 60x, 1.4 NA oil lens. In vitro assays were collected at 1 frame per 2.5 sec. Live cell movies were acquired at 1 frame every 2 sec for 82 or 42 sec, with 4-8 wells and 10-15 total fields per condition. Media was changed 5 hours before imaging and controls were performed on the same day for all conditions. Movies were processed in ImageJ (Schneider et al. 2012) and Fiji (Schindelin et al. 2012) including digital stabilization (Li 2008) and tracking of single cells. Fluorescent spots were automatically tracked using Imaris Software (Bitplane, Belfast, UK) with manual marking of the cell center. The resulting Microsoft Excel files for spot track parameters were compiled using software written for MatLab (Mathworks, Natick, MA), including calculation of track displacement towards the cell periphery (MT-D) using the cell center mark (Schafer et al. 2014;Toops et al. 2015). Final data and statistics were plotted and calculated in Graphpad Prism.

Immunostaining and image analysis
Indirect immunofluorescence was performed following conventional procedures. Briefly, cells were grown on coverslips, fixed in either cold methanol or 4% paraformaldehyde, blocked and permeabilized (1% BSA, 2% newborn calf serum and 0.01% Triton X-100), and then incubated with the primary and corresponding fluorophore-conjugated secondary antibodies as described previously (Bejarano et al. 2014). All slides were mounted for microscopy using Fluoromount-G (SouthernBiotech) containing DAPI (4′,6-diamidino-2-phenylindole) to highlight the nucleus.
Images were collected using Axiovert 200 fluorescence microscope (Carl Zeiss) equipped with a ×63 1.4 NA oil objective and ApoTome.2 system. All the images were prepared using Adobe Photoshop 6.0 software (Adobe Systems). For quantitative analysis, a single image was taken at the section of the maximum nucleus diameter and the number of fluorescent cytosolic particles (puncta) per cell and occupied area per total cell area were determined using the Analyze Particles function of ImageJ (NIH) after applying a fixed threshold to all images. All quantifications were done blindly. 3D reconstruction images were modelled as mixed rendering using the Inside4D module for AxioVision Rel. 4.8 after applying the Nyquist sampling criteria.
All representative images include quantification in the same or next panel and the number of repetitions is indicated in the corresponding figure legends. For organelle staining, cells were incubated with MitoTracker (50 mM) or Lysotracker (100 nM) (Invitrogen) for 20 min 37°C prior to fixation. In the case of isolated APGs, vesicles were incubated for 10 min at room temperature with primary antibodies, followed by incubation with fluorescence-conjugated secondary antibodies for an additional 10 min as previously described (Koga et al. 2011).

Immunoblot and electrophoresis
Subcellular fractions (20 μg of protein/lane) and homogenates (100 μg of protein/lane) from fed or 6-h-starved mice liver were subjected to immunoblot according to conventional procedures (Towbin et al. 1979) Briefly, samples were run on SDS-PAGE gels, transferred to nitrocellulose membranes, and, after blockage with low fat-milk, incubated with primary antibodies in 3% BSA.
Cell lysates were prepared by solubilization in RIPA buffer (1 % sodium deoxycholate, 0.1 % SDS, 0.15 M NaCl and 0.01 M sodium phosphate, at pH 7.2) containing protease and phosphatase inhibitors. The solubilized fraction was recovered in the supernatant after centrifugation at 12,000 g for 30 min, and protein concentration was measured by the Lowry method using bovine serum albumin (BSA) as a standard (Lowry et al. 1951). The proteins of interest were visualized by chemiluminescence using peroxidase-conjugated secondary antibodies in an LAS-3000 Imaging System (Fujifilm, Tokyo,Japan). Densitometric quantification was performed in unsaturated images using ImageJ (National Institutes of Health, Bethesda, MD).

Autophagy assays
Autophagic flux was measured by immunoblot analysis as changes in levels of autophagic markers (LC3-II and p62) upon inhibition of lysosomal proteolysis (Tanida et al. 2005).
Autophagosome content was evaluated as the number of fluorescent puncta after immunostaining with antibodies against endogenous LC3 (Kabeya et al. 2000). Monitorization of the mitochondrial delivery to lysosomes was performed by using the mitophagy reporter mt-Keima as described previously (Katayama et al. 2011). Quantification of fluorescence intensity of mt-Keima (Katayama et al. 2011) in GFP-channel (FL mito) and Rhodamine-channel (FLlyso) was used to calculate the mitophagy index as ratio of the intensity (FLlyso/FLmito). Ultrastructure analysis by electron microscopy was carried out as descried previously (Bejarano et al. 2014).
Briefly, culture cells were fixed in 4% paraformaldehyde/0.1% glutaraldehyde in 100mM sodium cacodylate, at pH 7.43, dehydrated and embedded in LR-White resin (LADD Research Industries). All grids were viewed on a JEOL 100CX II transmission electron microscope at 80 kV (JEOL, Peabody, MA). Morphometric analysis of transmitted electron micrographs was done blinded using ImageJ (NIH). Autophagic vacuoles were identified using standard criteria as previously (Bejarano et al. 2014). Briefly, vesicles were catalogued as autophagosomes or autolysosomes if they meet two or more for the following criteria: for autophagosomes they should have a double membrane (completely or partially visible), absence of ribosomes in the outer membrane, luminal density comparable to the surrounding cytosol and identifiable organelles or regions of organelles in their lumen; for autolysosomes they should have similar size but less than 40% of membrane visible as double, luminal density lower than surrounding cytosol and luminal material not recognizable as specific organelles and/or amorphous material.
Primary and secondary lysosomes (identified as single membrane vesicles of higher density and smaller average diameter) were excluded from the quantification. The term autophagic vacuole was used for those instances in which differentiation between autophagosomes and autolysosomes was not possible. In vitro fusion of autophagosomes and lysosomes in a microtubule-free system was measured as described before (Koga et al. 2010). Briefly, isolated APGs or lysosomes were incubated for 10 min at room temperature with primary antibodies against LC3 and LAMP2A, followed by incubation with fluorescence-conjugated secondary antibodies for an additional 10 min. Labeled vesicles were recovered by centrifugation and carefully resuspended in fusion buffer (10 mM HEPES, pH7; 10 mM KCl; 1.5 mM, MgCl2; 1 mM DTT; 0.25 M sucrose; and protease inhibitors) and were mixed in the reaction buffer (3 mM ATP, 2 mM GTP, 2 mM CaCl2, 0.16 mg/ml creatine phosphokinase, 8 mM phosphocreatine, and protease inhibitors) and incubated at 37°C for 30 min. The mixture was spotted on a glass slide, fixed with 8% formaldehyde in 0.25 M of sucrose for 15 min, and visualized under the fluorescence microscope as above.