Antibodies and plasmids
Mouse monoclonal antibodies to LC3 (NanoTools, Teningen Germany), phosphorylated NF-M/H (SMI-31) and unphosphorylated NF-M/H (SMI-32; Sternberger Monoclonal Inc., Lutherville, MD), dynein intermediate chain (DIC; Sigma, St. Louis, MO), β-tubulin (Sigma, St. Louis, MO), p62 (Abnova, Taiwan), ubiquitin (Millipore, Bedford, MA), microtubule-associated protein 2 (MAP2; Chemicon), rat monoclonal antibody to LAMP-2 (Hybridoma Bank, Iowa City, Iowa), rabbit polyclonal antibodies to LC3 ( Novus Biologicals, Littleton, CO), GFP (Abcam, Cambridge, MA), ubiquitin (Dako, Carpinteria, CA), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), guinea pig polyclonal antibody to p62 C-terminus (Progen Biotechnik) were purchased from commercial sources as listed above. Rabbit polyclonal antibodies against NF-L and cathepsin D were generated in our laboratory, mouse monoclonal anti-APP for human and murine APP referred to as antibody C1/6.1 has been described previously (Mathews et al., 2002
), and mouse monoclonal murine-specific APP antibody M3.2 that recognizes APP holoprotein, sAPPα, β-CTF, and Aβ was provided by Paul Mathews (Nathan Kline Institute, New York University, New York NY).
EGFP-LC3 was a gift from Dr. Noburu Mizushima (Tokyo Medical and Dental University, Tokyo, Japan); GFP-RFP-LC3 (Kimura et al., 2007
) was generously provided by Dr. Tamotsu Yoshimori (Osaka University). YFP-LAMP1 was provided by Dr. Joel Swanson (University of Michigan, Ann Arbor, MI). GFP-Rab7 was a gift from Dr. Anne Cataldo (McLean Hospital, Belmont, MA). DsRed-LC3 was made as described previously (Boland et al 2008
). pDsRed2-Mito and pAcGFP1-Endo were purchased from Clontech.
Cell culture and transfection
Embryonic mouse cortical neurons from C57B1/6J mice were cultured as described (Boland et al 2008
). Primary cortical neurons were harvested from embryonic day 16–17 pups. Pups were decapitated and cerebral cortices were removed and dissociated by incubation in Hibernating Medium (BrainBit) containing Papain (10 mg/ml) and DNase for 15 min at 37°C. Following digestion, cells were centrifuged at 1000 rpm for 3 min and the pellet was re-suspended in Modified Eagle’s Medium with 10% FBS (Hyclone) for counting and plating. Cells were plated (0.75 X 105
) on glass-bottom chamber dishes (BD Biocoat) or on circular coverslips (1.5 X 105
), both coated with poly-d-lysine (200μg/ml) and incubated at 37°C for at least 4 hours. After the cells attached, the plating medium was changed to Neurobasal medium containing B27, Penicillin/Streptomycin, and Glutamax. Half the volume of culture media was exchanged every 3 days. Penicillin/Streptomycin was removed from media after the first feeding. Neurons were transfected on DIV 3–4 using Lipofectamine 2000 (Invitrogen), based on the manufacturer’s suggestions with minor modifications. Conditioned media was replaced with fresh Neurobasal media containing 1–3 μg DNA/4μL of Lipofectamine 2000 in 500 mL Optimem (Invitrogen). The neurons were transfected for 30 minutes at 37°C, and subsequently washed 3 times with Neurobasal Medium to remove DNA and resuspended in conditioned media. Neurons were grown for 24 to 48 hours after transfection prior to treatments or time-lapse imaging.
For lysosomal inhibition or autophagy activation, cells were treated with leupeptin (20–100μM, International Peptides), E64 (10μM, Sigma), pepstatin (20μM, Sigma) or bafilomycin (10n–50nM, Sigma), or rapamycin (10nM, Sigma). For leupeptin-recovery in GFP-Rab7 neurons, transfected neurons were treated with leupeptin (20μM), or normal media (control recovery) for 24hours starting 6 hours after transfection and recovered in normal Neurobasal media for an additional 24 hours.
Western blot analysis
Neurons (5–7 DIV) grown in 6-well plastic dishes (BD Bioscience, Franklin Lakes, NJ) were washed (3X) in Tris buffered saline (TBS, pH 7.4) at RT, then scraped in 200ml/well of ice-cold lysis buffer (M-PER buffer containing Halt Protease Inhibitor Cocktail (1:100) and 50mM EDTA, Pierce, Rockford, IL). Protein concentration was determined using the Bradford Assay (Pierce) and samples were standardized to 1mg/ml using 70% trichloroacetic acid to precipitate cell lysates that were resuspended in equal volumes of lysis buffer. Sample loading buffer (2X) [62.5mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromophenol blue, 710mM β-mercaptoethanol] was added to cell lysates at a 1:1 ratio with lysis buffer before heating samples for 5 minutes at 90°C. Tris-glycine gels (Invitrogen) were loaded with 20 μg/well of protein and separated either on 4–20% gels or 16% gels to separate LC3-I and LC3-II. Separated proteins were transferred onto 0.2 μm nitrocellulose membranes (Protran, Whatman, Florham Park, NJ) for 24 hours at 10V. Membranes were rinsed in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) before being blocked at RT for 1 hour in a 5% non-fat milk/TBS-T solution. Membranes were incubated with primary antibody overnight at 4°C, washed three times in TBS-T and then incubated for 1 hour at RT in secondary antibody solution (3% non-fat milk/TBS-T) containing HRP-conjugated secondary antibodies (Promega, Madison, WI). Membranes were washed three times for 10 minutes in TBS-T before chemiluminescent substrates (ECL, Perkin Elmer) were applied and membranes were exposed to film. Densitometry of bands representing protein expression was performed using Image-J (NIH Image v.1.42l; http://rsb.info.nih.gov/ij
) software. For each immunoblot, the band intensity of each lane was normalized relative to the loading control and compared to the control lane. Subsequently, the percent change between treatments was calculated based on the normalized values.
Neurons were fixed for immunocytochemistry analyses by removing culture medium, washing (3x) in cold TBS and adding 4% paraformaldehyde/5% sucrose/TBS (pH 7.4) for 15 minutes at RT, washed 3 times with TBS and permeabilized for 15 minutes in 0.02% Triton-X/TBS. For LC3 and LAMP immunocytochemistry, cells were fixed in −20°C methanol for 5 minutes, washed 3X in TBS and permeabilized with digitonin/TBS (0.1 mg/ml). Non-specific antigens were blocked by incubation in 10% NGS/0.02% Triton-X/TBS blocking solution for 1 hour at RT. Primary antibodies were diluted in 0.5% BSA-TBS and incubated overnight at 4°C. The next day, cells were washed (3x) in TBS for 10 minutes prior to incubation with Alexa-488 or Alexa-546 secondary antibodies (1:1000 in blocking solution, Invitrogen) for 1 hour at RT. Neurons were washed (3x) in TBS for 10 minutes before mounting onto microscope slides with anti-fade Gelmount (Biomeda, Foster City, CA) and visualized using a Zeiss confocal microscope.
Magic Red, LysoTracker-Red, and Bodipy-Pepstatin loading
Neurons plated on 35mm glass-bottom chamber dishes (BD Biocoat) were incubated with 1μM of either BODIPY-pepstatin-FL (Invitrogen), Magic Red cathepsin B (Marker Gene Technologies) or Lysotracker Red (Invitrogen) in Neurobasal medium for 30–60 minutes at 37°C followed by washing with fresh Neurobasal Medium (2X). Subsequently, Neurobasal medium was replaced with pre-warmed Low Fluorescence Hibernating Medium (Brainbits, Springfield, IL) to reduce fluorescent background. For live imaging, cultures were placed in a humidified chamber maintained at 37°C and 5% CO2, and mounted on a Ziess LSM510 confocal microscope. Z-stacks were acquired using LSM 510 software.
35mm-glass bottom dishes (BD) containing normal growth medium (NB supplemented with B27) or Low Fluorescence Hibernate Medium (Brainbits) supplemented with B27, were mounted in a temperature-control stage on the confocal microscope, and maintained at 37°C and 5% CO2. The imaging was performed using a Zeiss confocal equipped with LSM 510 attachment using a 40X oil immersion lens. Laser lines at 488nm (GFP-tagged constructs, and YFP-construct) and 543 nm (DsRed-constructs) were used. Time-lapse recordings were acquired by scanning single plane images every 3–5 s for at least 5 minutes. Our data was collected in the axon (longest process emerging from the cell body) up to approximately 400 microns away from the cell body in any area where a substantial length of axon was in focus and appropriate to record movements. Kymographs of time-lapse movies were generated using ImageJ (Multiple Kymograph plugin).
Transport parameters of vesicle movement were generated using the ImageJ plugin MtrackJ (www.imagescience.org/meijering/software/mtrackj
). For each time-lapse movie, manually tracking individual vesicles using this program generates “Points” data (x, y, t-coordinates of each point/frame) as well as “Tracks” data (duration, min/max/mean V, etc.) based on an arbitrary reference point representing the most proximal point in the neurite that is closest in proximity to the cell body. After manually tracking vesicles until they were no longer visible, “Points” and “Tracks” data were transferred to MS Excel to calculate net transport direction, relative frequency of velocity, average velocity, and frequency of direction changes. Minimum velocity threshold was set at 0.1μm/s, where vesicles with maximum velocity less than 0.1 μm/s for the entire track were categorized as stationary, whereas vesicles with at least one movement with velocity of at least 0.1 μm/s were categorized as motile (moving). The transport direction was calculated based on the algebraic sign of dx/dt from frame to frame (x1−x0)/(t1−t0), where anterograde movement was positive and retrograde movement was negative. Net transport direction was determined by comparing the initial and final (x, y) position of the vesicle, and classified as retrograde, anterograde or stationary (if no movement above 0.1 μm/s occurred despite a net change in position). Motility and net transport direction was expressed as an averaged percent of total vesicles per axon. The relative frequency of instantaneous velocity was obtained by sorting and binning instantaneous velocity values of each track in intervals of 0.2μm/s, and using the COUNT function of MS Excel for each bin divided by the total number of movements. Frequency of direction changes were calculated using an MS Excel function created by the author to calculate the total number of switches in a given track divided by the total duration of the track. A switch is defined as different direction of motile behavior from one frame to the next frame. All values represent means with standard error of the means (sem). Student’s t-test for statistical analyses (p values) was performed in MS Excel.
Morphometric Analysis of GFP-LC3 neurons
The number of GFP-LC3 neurons containing GFP-puncta in the cell body or in neurites, were counted using confocal images of GFP-LC3 transfected neurons, imaged at 40X direct magnification for the various treatment conditions. At least 40 neurons were examined and values are expressed as a percent total of counted cells. Based on visual detection of puncta, we categorized neurons into as either neurons with vesicular structures or neurons with no puncta (diffuse LC3 only). When punctate LC3 was detectable, the number of cells where puncta was visualized in the cell body, axons, or both was counted. All numerical values are expressed as a mean +/− SEM. For counting number of GFP-LC3 vesicles in axons, the number of LC3 vesicles were manually counted and subsequently divided by the total length of the axon as measured using the NIH ImageJ plugin, NeuronJ.
The number of GFP-LC3 vesicle swellings per length of neurite and the percentage of GFP-LC3 neurons containing swellings were analyzed by measuring the total length of the GFP-LC3 neurite in the 40X field using ImageJ plugin, NeuronJ, and counting the number of visible swellings. At least 30 neurons were examined for each condition.
All animal studies were carried out according to the regulations of the IACUC at the Nathan Kline Institute. Transgenic mice expressing either the Swedish mutation of human APP (APPK670M/N671L; referred to as APP) or APPswe/PS1M140L (referred to as PSAPP) at the indicated plaque-bearing ages were anesthetized and transcardially perfused with 4% paraformaldehyde/0.1M sodium cacodylate buffer, after which brains were dissected and submerged in 2% glutaraldehyde/0.1 M sodium cacodylate buffer overnight at RT. 50μm coronal sections cut by vibratome were postfixed in 1% osmium tetroxide in Sorensen’s phosphate buffer for 1 hour at RT and dehydrated in a series of increasing concentration of ethyl alcohols (50–100%). For neurons, DIV 4–5 neurons grown on glass coverslips were fixed by removing culture medium, washing once in 37°C supplement-free Neurobasal medium and adding 4% paraformaldehyde/1% glutaraldehyde/5% sucrose in 0.1M sodium cacodylate buffer (pH 7.2; Electron Microscopy Sciences (EMS), Hatfield, PA) for 24 hours at RT. Following fixation, neurons were washed (x3) in 0.1M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide and progressively dehydrated in a graded series of ethanols (50% – 100%) followed by embedding in Epon (EMS, Fort Washington, PA) for at least 3 days at RT. Tissue was embedded in Epon were cut serially into ultrathin (0.06 μm) sections. Ultrathin sections were stained with uranyl acetate and lead citrate. Ultrathin sections were cut from Epon-embedded blocks and placed on copper grids for structural analysis.
Quantitative analysis of AVs from EM images
For quantification of organelles in neuritic swellings after leupeptin treatment, 30 randomly selected EM images with accumulated organelles were captured at a final magnification of 10,500×, and the number of each type of AV, mitochondria and single membrane vesicles was counted using the criteria for AV identification previously established (Nixon et al., 2005
). For APP and PSAPP mice, the same procedure was applied, but 50 dystrophic neurites in each of two mice per genotype were used at the indicated ages.