GFP-LC3 transgenic mice, strain name B6.Cg-Tg(CAG-EGFP/LC3)53Nmi/NmiRbrc, were developed by N. Mizushima (Tokyo Medical and Dental University, Tokyo, Japan; Mizushima et al., 2004
) and deposited into the RIKEN BioResource Center in Japan. Transgenic mice overexpressing SOD1G93A
, strain name B6SJL-Tg(SOD*G93A)1Gur/J, were purchased from The Jackson Laboratory. Constructs include DsRed2-mito (gift from T. Schwarz, Harvard Medical School, Boston, MA), monomeric RFP-Ub (Addgene), LAMP1-RFP (Addgene), mCherry-EB3 (gift from I. Kaverina, Vanderbilt University Medical Center, Nashville, TN), GFP-Rab5 (gift from M. Zerial, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany), GFP-Rab7 (Addgene), GPP130-mCherry (gift from A. Linstedt, Carnegie Mellon University, Pittsburgh, PA), and mCherry-EGFP-LC3 (gift from T. Johansen, University of Tromsø, Tromsø, Norway; Pankiv et al., 2007
, DIC1B, and DIC2C (gifts from K. Pfister, University of Virginia, Charlottesville, VA), Kif5C tail (gift from M. Setou, Hamamatsu University School of Medicine, Shizuoka, Japan), Kif3A (gift from K. Kaibuchi, Nagoya University Graduate School of Medicine, Nagoya, Japan), and the CC1 domain (E216-Q550) of p150Glued
were recloned into pmCherry (Takara Bio Inc.). Antibodies include a polyclonal antibody against LC3B (Abcam) and monoclonal antibodies against dynein intermediate chain (clone 74.1; Millipore), p150Glued
(BD), kinesin-1 heavy chain (clone H2; Millipore), kinesin-2 (clone K2.4; Abcam), and SOD1 (Sigma-Aldrich).
GFP-LC3 × SOD1G93A cross
GFP-LC3 transgenic mice were crossed with SOD1G93A transgenic mice in our animal facility. DRG neurons were isolated at early stage disease (84 d) and late-stage disease (mean of 125 d). Late-stage disease was determined based on the criteria established by The Jackson Laboratory (score of 3 in the neurological scoring system). Age-matched littermates served as controls. The Institutional Animal Care and Use Committee at the University of Pennsylvania approved all animal protocols.
Live-cell imaging of DRG neurons
DRG neurons were isolated according to Perlson et al. (2009)
and maintained in F-12 media (Invitrogen) with 10% heat-inactivated fetal bovine serum, 2 mM l
-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For live-cell analysis, DRG neurons were plated on glass-bottom dishes (World Precision Instruments, Inc.) and cultured for 2 d at 37°C in a 5% CO2
incubator. If necessary, before plating, neurons were transfected with 0.5 µg plasmid DNA using a Nucleofector (Lonza) according to the manufacturer’s specifications. Imaging was performed in low fluorescence nutrient medium (Hibernate A; BrainBits) with 2% B27 and 2 mM GlutaMAX. For LysoTracker red labeling, DRGs were incubated in 100 nM LysoTracker red (Invitrogen) in F-12 culture medium for 30 min at 37°C in a 5% CO2
incubator and washed twice in culture medium before imaging.
For GFP-LC3 motility and particle tracking analysis (; ; Fig. S1, C and D; and Fig. S3) as well as mCherry-EGFP-LC3 analysis (), imaging was performed on an inverted epifluorescence microscope (DMI6000B; Leica) using an Apochromat 63×, 1.4 NA oil immersion objective (Leica) in an environmental chamber at 37°C. Digital images were acquired with a charge-coupled device camera (ORCA-R2; Hamamatsu Photonics) using LAS-AF software (Leica). Images were taken once every 3 s for a total of 3 min. For dual-color videos, images were taken consecutively, with green followed by red.
For the biogenesis experiments (; and Fig. S1 B) and colocalization experiments (; ; ; Fig. S1 A; and Fig. S2), imaging was performed on a spinning-disk confocal (UltraVIEW VoX; PerkinElmer) with a microscope (Eclipse Ti; Nikon) with the Perfect Focus System using an Apochromat 100×, 1.49 NA oil immersion objective (Nikon) in an environmental chamber at 37°C. Digital images were acquired with an EM charge-coupled device camera (C9100; Hamamatsu Photonics) using Volocity software (PerkinElmer). For biogenesis experiments, images were taken once every 2, 3, or 5 s for 10–20 min. Photobleaching of GFP-LC3 was achieved using a 488-nm laser at 100% power for 15 iterations. The GFP-LC3 signal was bleached completely throughout the depth of the neurite. For colocalization experiments, images were taken once every 2 s for 5 min, with green followed by red. Photobleaching of mCherry-SOD1G93A and RFP-Ub was achieved using a 561-nm laser at 100% power for 30 iterations. Recovery images were taken once every 2 s for 5 min. Images were assembled using ImageJ (National Institutes of Health) and Photoshop (Adobe).
GFP-LC3–positive puncta that moved a net distance ≥10 µm were manually tracked frame to frame for the duration of the entire video (3 min) using the particle tracking function in MetaMorph (Molecular Devices). A mean vesicle velocity was calculated by averaging all frame to frame instantaneous velocities (excluding paused values) for a single puncta. A pause was defined as a single or consecutive instantaneous velocity value of <0.067 µm/s, empirically determined to be the resolution of our system. A reversal was defined as a single or consecutive instantaneous velocity value of >0.067 µm/s in the opposite direction as compared with the net displacement of the vesicle. The number of reversals within 100 µm was determined based on the net distance a vesicle traveled during the 3 min.
Kymographs were generated using MetaMorph from neurites having at least one GFP-LC3–positive puncta that traveled a net distance of ≥10 µm. From each kymograph, the percentage of autophagosomes moving in the net retrograde direction (≥10 µm) versus net anterograde direction (≥10 µm) was determined. Nonprocessive vesicles that did not move a net distance of 10 µm exhibited bidirectional and stationary motility. From these kymographs, the total number of vesicles was determined and normalized by kymograph length (micrometers). Flux (number of vesicles moving within 100 µm/min) was determined by the sum of retrograde and anterograde vesicles (excluding bidirectional/stationary vesicles) and normalized by kymograph and video length. For mCherry-EGFP-LC3 analysis, the number of LC3 puncta positive for both GFP and mCherry fluorescence was counted based on kymographs. The proximal region of the neurite was defined as being within ~200 µm of the cell body, and the distal region was within ~100 µm of the end of the neurite.
Fractions enriched for autophagosomes were prepared from brains of GFP-LC3 transgenic mice using protocols adapted from Morvan et al. (2009)
and Strømhaug et al. (1998)
. Two brains were homogenized in 10 ml of 250 mM sucrose in 10 mM Hepes-KOH, pH 7.4 (with 1 mM EDTA for three-step gradient protocol) using a 30-ml homogenizer with a round-bottom Teflon pestle. Volumes of the gradient steps were scaled proportionately for a rotor (SW41; Beckman Coulter). The final gradient of the three-step fractionation protocol (Strømhaug et al., 1998
) was spun in a rotor (TLS-55; Beckman Coulter). Equal total protein of low speed supernatant and the autophagosome-enriched fraction was separated by SDS-PAGE and subjected to immunoblot analysis.
For immunofluorescence analysis, DRG neurons were plated on coverslips and cultured for 2 d. Cells were washed once in PBS (150 mM NaCl in 50 mM NaPO4, pH 7.4) and fixed in 3% PFA in PBS for 15 min at room temperature. Cells were washed twice in PBS and blocked and permeabilized in 2% (wt/vol) BSA and 0.1% (wt/vol) saponin in PBS for 1 h. All subsequent steps were performed in blocking/permeabilization buffer. Samples were incubated in primary antibody for 1 h, washed 3 × 5 min, incubated in secondary antibody for 1 h, washed 3 × 5 min, and mounted with ProLong gold.
Online supplemental material
Fig. S1 shows the anterograde movement of EB3 in DRG axons, FRAP of GFP-LC3 along the axon, and distributions of mean vesicle velocities and percentage of pausing for GFP-LC3 puncta along the axon. Fig. S2 shows that autophagosomes along the axon are positive for DIC2C-mCherry and mCherry-Kif3A but not the Golgi marker GPP130-mCherry. Fig. S2 also shows that LysoTracker red–positive compartments are positive for the late endosomal marker Rab7 but are largely negative for the early endosomal marker Rab5. In Fig. S3, we present data showing that despite the accumulation of SOD1G93A
aggregates along the axon, autophagosome motility, direction, velocity, density, and flux are unaffected in a mouse model of fALS. Video 1 shows the appearance and growth of GFP-LC3–positive puncta in the neurite tip that grow into ringlike structures characteristic of autophagosomes. Video 2 shows an autophagosome escaping from the bidirectional pool at the neurite tip and moving processively toward the cell soma. Video 3 shows the robust retrograde motility of autophagosomes along the axon, and Videos 4 and 5 show that dynein co-migrates with these autophagosomes transfected with DIC1B-mCherry (Video 4) or DIC2C-mCherry (Video 5). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201106120/DC1