CamK-Cre transgenic mice, Dat
mice, hAPP-Tg and tau KO mice, used in this study were generated previously
[19, 20, 37, 60–62]. CamK-Cre Tg and tau KO mice were purchased from Jackson Laboratories. All animals were maintained in the animal facility of the Columbia University Medical Center. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Genomic DNA extracted from mouse tails was amplified by PCR for genotyping using standard methods. The PCR primers are the followings: 5’-AGA TGT TCG CGA TTA TC-3’, 5’-AGC TAC ACC AGA GAC GG-3’ for Cre transgene; 5’-TGC TCT GTG AAC TGC CCT GTT T-3’, 5’-TGT TCC TGT GCA CTG CCT CAT T-3’ for Atg7 wild-type allele; 5’-CTT GGG TGG AGA GGC TAT TC-3’, 5’-AGG TGA GAT GAC AGG AGA TC-3’ for Atg7 floxed allele.
Mice were perfused and fixed in 4% paraformaldehyde and post-fixed at 4°C overnight, 50 μm coronal brain sections were generated using a vibratome. The sections were blocked with PBS containing 5% normal donkey serum [NDS], 0.2% Triton X-100 [Tx] for 1 h, and incubated with the solution (PBS, 1% NDS, 0.2% Tx) containing primary antibody at 4°C overnight. The following antibodies were used; anti-TH (P60101, Pel-Freez), anti-TuJ1 (MMS-435P, COVANCE), anti-MAP2 (AB5622, Millipore), anti-cleaved caspase-3 [Asp175] (#9661, Millipore), anti-active caspase-3 (AB3623, Cell Signaling Technology), anti-ubiquitin (Sigma-Aldrich), anti-p62 (03-GP62-C, American Research Products), anti-Aβ [4G8] (SIG39200, COVANCE), anti-Aβ [6E10] (SIG39300, COVANCE), anti-αSynuclein (610786, BD Bioscience) (AB5038, Millipore) (ab1903, ab24715, Abcam), anti-phosph-tau TG3 and PHF1 (gifts from Dr. Peter Davies, Alberts Einstein College of Medicine), anti-phospho-tau AT8, AT100, AT180, and AT270 (Pierce), anti-total GSK3β (#9315, Cell Signaling Technology), anti-phospho-GSK3α/β [Y279/Y216] (ab52188, Abcam), anti-phospho-GSK3β [S9] (ab30619, Abcam), anti-total CRMP2 (#9393, Cell Signaling Technology), anti-phospho-CRMP2 [T514] (#9397, Cell Signaling Technology), anti-Cdk5 (MAB5410, Millipore), anti-p35/25 (#2680, Cell Signaling Technology), anti-β-catenin (#9581, 9587, Cell Signaling Technology), and anti-β-catenin (#610154, BD Biosciecnes). For secondary detection, Cy3- or FITC-conjugated antibodies were incubated for 1 h (Jackson ImmunoResearch). Photographs were taken using a Zeiss LSM 510 Meta confocal microscope.
To obtain neuronal cell count, 50 μm coronal brain sections were made using a vibratome. In order to count CA1 neurons, the first 30 sections from the rostral hippocampus were stained with rabbit anti-MAP2 antibody (AB5622, Millipore) at a dilution of 1:500, as well as NeuroTraceTM Fluorescent Nissl stain (N21480, Invitrogen). MAP2-positive neurons were visualized using a Cy3-conjugated secondary antibody (Jackson ImmunoResearch). MAP2 and Nissl double-positive neurons in the CA1 regions were counted manually. In order to count TH-positive neurons, sections covering the entire substantia nigra (25-30 sections / mouse) were stained with sheep anti-TH antibody (P60101, Pel-Freez) at a dilution of 1:250. TH-positive neurons were visualized using the ABC Kit (PK6106, Vector Laboratories) and DAB (SK4100, Vector Laboratories). TH-positive neurons in the substantia nigral regions were counted manually under the light microscope.
Electron microscopic analysis was as described
. Anesthetized mice were perfused and fixed in PBS containing 4% paraformaldehyde and 0.5% gultaralaldehyde. The brains were post-fixed at 4°C for 2 h, and the 80 μm vibratome sections were made. The sections were treated in 1% osmium tetroxide, then dehydrated in pure ethanol and infiltrated overnight with Epon 812. Epon was polymerized at 60°C for 24 h, cooled and embedded in a larger Epon capsule. Ultrathin sections were cut with an MT5000 ultramicrotome, stained with uranyl acetate and lead citrate. Images were taken with a JOEL 100S Electron Microscope (JOEL USA).
Preparation of soluble and insoluble fractions was performed as described with some modifications
. Cortical and hippocampal tissues from mouse brains were homogenized in 5× volume of ice-cold 0.25M sucrose buffer (50mM Tris-HCl [pH7.6]) containing protease inhibitors (P8340, Sigma) and phosphatase inhibitors (#78420, Thermo Scientific). The homogenized tissues were centrifuged at 500× g for 10 min at 4°C. The supernatants were lysed with an equal volume of cold sucrose buffer containing 1% Triton X-100. The lysates were centrifuged at 13,000× g for 15 min at 4°C. The supernatants contained the soluble fraction. The pellets were resuspended in 1% SDS in PBS (insoluble fraction). Both fractions were subjected to standard Western Blotting analysis. The antibodies used here are: anti-phospho-tau AT8, AT100, AT180, AT270, TG3 and PHF1, anti-Tau1 and anti-Actin (ab3280, Abcam). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and SuperSignal West Pico or Dura (#34077, 34075, Pierce) were used for detection.
Brains from CamK-Atg7 cWT and cKO mice littermates (~12 weeks of age) were quickly removed and transverse hippocampal slices (400 μm) were isolated with a Leica VT1200 Vibratome (Leica, Bannockburn, IL), and placed in ice-cold cutting solution (CS: 110 mM Sucrose, 60 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 28 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, 5 mM Glucose, 0.6 mM Ascorbate. Slices were placed in an interface chamber (Scientific Systems Design, Mississauga, Ontario, Canada) and maintained at 32°C in ACSF (2 ml/min) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2. All solutions were constantly caboxygenated with 95% O2 + 5% CO2. Slices were allowed to recover for 120 min on the electrophysiology rig prior to experimentation. Bipolar stimulating electrodes (92:8 Pt:Y) were placed at the border of area CA3 and area CA1 along the Schaffer-Collateral pathway. ACSF-filled glass recording electrodes (1–3 MΩ) were placed in stratum radiatum of area CA1. Basal synaptic transmission was assessed for each slice by applying gradually increasing stimuli (0.5–15V), using a stimulus isolator (A-M Systems, Carlsborg, WA) and determining the input:output relationship. All subsequent stimuli applied to slices was equivalent to the level necessary to evoke a fEPSP that was ~40% of the maximal initial slope that could be evoked. Synaptic efficacy was continuously monitored (0.05 Hz). Sweeps were averaged together every 2 min. fEPSPs were amplified (A-M Systems Model 1800) and digitized (Digidata 1440, Molecular Devices, Sunnyvale, CA) prior to analysis (pClamp, Molecular Devices, Sunnyvale, CA). Stable baseline synaptic transmission was established for 30 min. Slices were given high-frequency stimulation (HFS) to induce long-term potentiation (LTP) using one train of 100 Hz for one second. Stimulus intensity of the HFS was matched to the intensity used in the baseline recordings. fEPSP initial slopes from averaged traces were normalized to those recorded during baseline. Two-way RM-ANOVA were used for electrophysiological data analysis with p < 0.05 as significance criteria.
10-13-mon-old male CamK-Atg7 cWT or CamK-Atg7 cKO mice were used (n = 8 - 10). The mice were placed in a conditioning chamber (Med Associates) for 2 min before the onset of a tone (conditioned stimulus) (30 s, 85 dB sound at 2800 Hz) and conditioned by a single electrical foot shock (0.45 mA) in the last 2 s. The mice were left in the chamber for another 30 s and placed back into their home cage. Contextual fear learning was measured in the same chamber 24 h after the training by monitoring the freezing for 5 min without electrical shock. Cued fear learning was measured 24 h after the contextual testing. The mice were placed in a novel chamber for 2 min (pre-conditioning). After that, the mice were exposed to the conditioned stimulus for 3 min, and the freezing was monitored. Freezing behavior was scored using FreezeView software (Med Associates Inc.).
Five-week-old Dat-Atg7 cWT and Dat-Atg7 cKO mice were treated with Alsterpaullone (A1136, A.G. Scientific)
. The drug was dissolved in saline containing 20% DMSO/ 25% Tween80, sonicated, and injected intraperitoneally at a dose of 5 mg/kg every day for 3 weeks. After the final injection, the mice were perfused and processed for histological analyses. We used Dat-Atg7 cWT mice as controls for Dat-Atg7 cKO mice, to address potential phenotypes due to Cre transgene inserted at the DAT locus
All comparisons between groups were made using the Mann-Whitney U-test (for two samples) or non-repeated measures ANOVA (for multiple samples). The values are expressed as the means ± S.E. A p value less than 0.05 is considered significant.