Cell Culture and Reagents
Primary cultures of embryonic rat hippocampal neurons were prepared as described (Kaech and Banker, 2006
). Hippocampal neurons from BDNF knockout mice were obtained from crossing BDNF +/− mice (Ernfors et al., 1994
). The two hippocampi from each 14–15-d embryo were put in separate tubes; a piece of cortex from each embryo, for later determination of genotype with BDNF specific primers, was also put in separate tubes. The hippocampi were then trypsinized and dissociated as for the rat hippocampal cultures, followed by plating in individual dishes. Each dish was later classified as BDNF +/+, +/−, or −/−, according to the PCR analysis of the corresponding cortical specimens. For biochemical analysis, 105
cells were plated into 3-cm plastic dishes coated with poly-l
-lysine (0.1 mg/ml) and containing minimal essential medium with N2 supplements (MEM-N2). Neurons were kept under 5% CO2
at 37°C. Where indicated, cells were incubated with particular inhibitors for 2 h to assess target inhibition or for 36 h to measure cell death. The monoclonal anti-Flotillin 1 antibody, clone 18, was from Transduction Laboratories (Franklin Lakes, NJ); the polyclonal chicken anti-BACE 1 antibody, raised against Fc-Asp 2-fusion protein, and the monoclonal anti-transferrin receptor antibody were from Zymed Laboratories (San Francisco, CA). Antibodies used to detect pTrk were polyclonal rabbit either from Santa Cruz Biotechnology (Santa Cruz, CA) or from Cell Signaling (Beverly, MA). Antibodies used to detect TrkB were polyclonal rabbit either from Santa Cruz Biotechnology or from BD Transduction Laboratories (Lexington, KY). Mouse monoclonal anti-tubulin were all from Cell Signaling, and the rabbit antiserum anti-p85 was from Upstate Biotechnology (Lake Placid, NY). CYP46A1 antibody was provided by Dr. Russell (University of Texas Southwestern Medical Center, Dallas, TX) and described previously (Lund et al., 1999
Stimulation by Ligand and Neurotrophin Inhibition
Predifferentiated 10 days in vitro (10 DIV) hippocampal neurons were cultured in a serum-free medium and stimulated in independent experiments with 100 ng/ml BDNF for 30 min, 50 ng/ml nerve growth factor (NGF) for 10 min, or with a conditioned medium derived from 26 DIV neurons for 120 min. Neurotrophin activities were neutralized in 10 DIV cell culture medium by adding agonist antibodies raised against BDNF, NGF, NT3, NT4/5 (anti-neurotrophin antibody sample pack 1; Chemicon, Temecula, CA) at a concentration of 5 mg/ml for 120 h.
Plasma Membrane Purification
Mature rat hippocampal neurons were lysed by osmotic swelling in 25 mM MES Buffer, pH 7.0, 5 mM dithiothreitol, 2 mM EDTA and protease inhibitor cocktail (Roche, Indianapolis, IN), in ice for 15 min. Extracts were homogenized with a syringe (22 gauge) and centrifuged for 5 min at 700 × g and at 4°C. Supernatants were brought to a final concentration of 1.6 M sucrose, put on the bottom of an SW50 rotor centrifuge tube (Beckman Instruments, Fullerton, CA), overlaid with a continuous sucrose gradient from 1.6 to 0.4 M, and subsequently centrifuged at 12,0000 × g for at least 12 h at 4°C. Eight fractions were collected from the top to the bottom of the tube. The fractions that contain plasma membrane were subjected to centrifugation at 10,0000 × g for 18 h at 4°C. The fractions were finally analyzed by Western immunoblot using antibodies against specific markers of membrane compartments.
Separation of DRMs
Detergent-resistant membranes (DRMs) were prepared from hippocampal membranes (see above) by Triton X-100 extraction, and soluble and insoluble (DRMs) fractions were separated as described previously (Tansey et al., 2000
). For some experiments, total plasma membrane extracts were brought to 60% sucrose in MES Buffer Saline (MBS), and a sucrose step gradient was overlaid (35 and 5% sucrose). After centrifugation at 100,000 × g
for 18 h at 4°C, fractions were collected from the top of each tube. Fractions 4 and 5 were identified as the DRM fractions by the presence of the DRM marker Flotillin 1.
Western Blotting, Immunoprecipitations, and Antibodies
Rat or mouse hippocampal tissues were homogenized in PBS containing 9% sucrose, protease inhibitors (CLAP: pepstatin, antipain, and chymostatin, each at a final concentration of 25 mM) and 1 mM sodium orthovanadate using a dounce homogenizer and 10 passages through a 22-gauge syringe. Samples were centrifuged for 10 min at 2500 × g, and supernatants were considered as total extracts. A further centrifugation step was performed at 10,000 × g for 1 h at 4°C to pellet the membrane fraction.
Total and membrane pellets of hippocampal neurons were extracted with two different detergents depending on the aim of the experiment: Nonidet P-40 (1% Nonidet P-40, 10% glycerol, 100 mM NaCl, 2 mM EDTA, 10 mM Tris/HCl, 500 mM sodium orthovanadate, and protease inhibitors) or Triton X-100 buffer (100 mM MES, pH 7, 150 mM NaCl, 1% Triton X-100) in the presence of 1 mM sodium orthovanadate and protease inhibitors. Extracts were clarified by centrifugation, and the protein concentrations were quantified by the BCA method (Bio-Rad Laboratories, Hercules, CA). Proteins were then transferred onto nitrocellulose membranes and probed with primary antibodies for 16 h. Species-specific peroxidase-conjugated secondary antibodies were subsequently used to perform enhanced chemiluminescence detection (Amersham, Little Chalfont, United Kingdom).
For immunoprecipitations, hippocampal neurons were extracted as described above. The soluble and insoluble fractions were diluted with the appropriate buffer to equalize detergent amount (1% Triton and 0.1% SDS). For TrkB and p85 immunoprecipitations, 1 μg of polyclonal antibody/sample was added to protein A–Sepharose beads, and samples were rotated at 4°C overnight. Beads were washed with cold lysis buffer. Immunoprecipitated complexes were separated by 10% PAGE-SDS electrophoresis and subjected to Western blot analysis. Quantification was done by densitometry of the autoradiograms using the NIH Image J software package (http://rsb.info.nih.gov/ij/
Lipid Extraction and Thin-Layer Chromatography
Lipids were extracted from plasma membranes obtained from rat hippocampal tissues or cultured neurons according to Bligh and Dyer (1959)
. Extracted lipids (cholesterol, ceramide, and sphingomyelin) were subsequently analyzed by thin-layer chromatography (TLC) on silica gel 60 HPTLC plates using a double-step system (hydrophilic running solvent: chloroform/acetone/acetic acid/methanol/water 50:20:10:10:5) and hydrophobic solvent: hexane/ethyl acetate (5:2) for cholesterol and ceramide. The plate was then dried and developed by spraying with 7% sulfuric acid and heating at 150°C in an oven.
Membrane Cholesterol Quantification, Reduction, and Replenishment
Total cholesterol was measured in membrane extracts from hippocampal neurons in vitro or whole hippocampus as previously described (Abad-Rodriguez et al., 2004
). For the induction of membrane cholesterol reduction, 0.4 μM mevilonin and 0.5 mM β-methyl-cyclo-dextrin were added daily to 5-d-old neurons during 4 d (96 h). At the end cholesterol was measured, to ascertain that the treatment did not result in more than 25% reduction. Only validated cases membranes were utilized for further experimentation. Cells were scraped in MES Buffer-CLAP at 4°C, and centrifuged at 1000 × g
. Postnuclear supernatants were further centrifuged at 10,0000 × g
for 1 h at 4°C to get the membrane pellet. Protein and cholesterol concentrations were measured after resuspension in MES Buffer-CLAP with 0.1% Triton X-100.
Cholesterol-MCD inclusion complexes were prepared as described (Klein et al., 1995
). These complexes, containing 0.3 mM cholesterol, were added to the medium at a final concentration of 0.3 mM together with 2 mg/ml free cholesterol. The treatment was performed for 15 min, and then cells were washed and scraped with MES Buffer-CLAP 0.1% Triton X-100 at 4°C
Analysis of Gene Expression
Total RNA samples were prepared from 5 × 105 hippocampal neurons at 10, 15, 20, and 25 DIV using the ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA). One hundred nanograms of total RNA were first incubated with poly-T oligonucleotides and subjected to reverse transcription using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA), following the manufacturer's instructions. cDNA was used as a template for the real-time PCR analysis based on the SYBR Green PCR Master Mix assay with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Real-time PCR primers were designed using primer-Express software (Applied Biosystems). Expression of genes encoding TrkA, TrkB, and BDNF and of genes involved in cholesterol synthesis and metabolism was analyzed, and transcriptional expression was normalized using the housekeeping gene encoding for the β Actin (actb) as reference in order to avoid differences due to possible RNA degradation or different reverse transcription efficacy. Relative expression levels of the targets were calculated with the comparative cycle threshold (CT) method according to the following formula: sample ΔCT = (gene CT − actbCT). To follow expression during ageing, results were normalized to a calibration standard sample of RNA (10 DIV sample). This control was assigned the normalization ratio = 1 and the normalization ratio of each sample was calculated according to the formula: 2−ΔΔCT = 2−(sample ΔCT − calibrator ΔCT).
For the light microscopy detection of Cyp46, neurons on glass coverslips were washed in PBS (0.01M), fixed with 4% paraformaldehyde, and processed for immunofluorescence by using anti-CYP46. For the analysis of phospho-TrkB on the surface of young and old cells, cells were first incubated with the corresponding antibody (see above) at 15°C for 15 min to minimize loss by internalization, and excess antibody washed away and then fixed and processed for immunofluorescence microscopy. The surface distribution of the ganglioside GT1b was detected with anti-GT1b mAb (Seikagaku, Tokyo, Japan). Samples were analyzed on an Olympus IX81 fluorescence microscope (Melville, NY). Quantification was performed using the NIH ImageJ software, and signal intensities were normalized by area.
targeting sequence was designed by using the RNA interference design algorithm at www.dharmacon.com/DesignCenter/DesignCenterPage.aspx
, and 46-sense and 46-antisense oligos encoding cyp46A1
-short hairpin RNA (shRNA) were designed as reported by Rubinson et al. (2007)
. To generate pSi46 plasmid, these oligonucleotides were annealed and cloned into the HpaI and XhoI sites from pLentiLox 3.7 (Rubinson et al., 2007
): 46-sense oligo: 5′-TAGATGTACCGTGCGATTCATTCAAGAGATGAATCGCACGGTACATCTTTTTTTC-3′; 46-antisense oligo: 5′-TCGAGAAAAAAAGATGTACCGTGCGATTCATCTCTTGAATGAATCGCACGGTACATCTA-3′.
The full ORF expression clone p46+ including the full cyp46A1 mouse open reading frame was purchased from RZPD (German Resource Center for Genome Research).
Primary dissociated hippocampal cells isolated from rat embryos (on embryonic day 18) were transfected using the Rat Neuron Nucleofector Kit from Amaxa Biosystems (Köln, Germany).