Generation of shRNA constructs for APP and Dab1 (Young-Pearse et al., 2007
), and control shRNA (Young-Pearse et al., 2007
)and DISC1 (Kamiya et al., 2005
), were previously described and characterized. The generations of the following constructs were previously described: HA-tagged human DISC1-L, -Lv, 1-402, 403-854, DISC1ΔNDEL1 (DISC1 lacking the NDEL1 binding site) and DISC1ΔSA (DISC1 lacking a self-association site), HA-tagged mouse DISC1, and NDEL1-myc (Kamiya et al., 2005
), Itgβ1-FLAG, FLAG-APP, and APLP1-FLAG (Young-Pearse et al., 2008
), FLAG-APP ΔCT, and APPNATA (Young-Pearse et al., 2007
) and NPR3-FLAG (Hemming et al., 2008
). APP666 encodes human APP from amino acids 1 to 666 and APP680 encodes amino acids 1-680 (695 splice variant numbering for both). DISC1-S and –Es were cloned by PCR and sequenced to confirm amino acid identity with entries for these human DISC1 splice variants in Genbank (NCBI).
In utero electroporation
Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were housed and cared for under the guidelines established by Harvard University’s Institutional Animal Care and Use Committees in compliance with federal standards. Timed pregnant female rats embryonic day 15.5 (E15.5) were anesthetized with ketamine/xylazine (100/10 mixture, 0.1 mg/g body weight, i.p.). The uterine horns were exposed, and a lateral ventricle of each embryo (both amles and females) injected with DNA constructs and Fast Green (2 mg/ml; Sigma, St. Louis, MO) via a microinjector (Picospritzer III; General Valve, Fairfield, NJ) and pulled glass capillaries. For characterization of shRNA phenotypes, 1.0 –1.5 mg/ml of shRNA was co-electroporated with 0.5 mg/ml pCAG-green fluorescent protein (GFP). For rescue experiments, 0.5 mg/ml shRNA was co-electroporated with 0.5 mg/ml pCAG-GFP and 3.0 mg/ml rescue constructs. All rescue constructs were expressed in the pCAGGs vector. Electroporation was accomplished by discharging a 500 mF capacitor charged to 50–100 V with a sequencing power supply or with a BTX square wave electroporator, at 50–75 V, for 50 ms on followed my 950 ms off for 5 pulses. The voltage was discharged across copper alloy oval plates placed on the uterine wall across the head of the embryo. Brains from rat embryos were harvested 72 hours following electroporation in 4% paraformaldehyde by immersion. For each plasmid combination, at least three independent brains were analyzed. For BrdU studies, 1 ml of a 10 mg/ml BrdU solution was injected intraperitoneally 24 hours after in utero electroporation.
Cell lines and Western blot analysis
COS cells (monkey kidney cell line) were transiently transfected using Lipofectamine 2000 (Invitrogen) in 10-cm dishes in duplicate with listed constructs. Cells were lysed after 48–72 h in 1% Nonidet P-40 (NP-40) STEN buffer [150 mM sodium chloride, 50 mM Tris, 2 mM EDTA, and 1.0% (v/v) NP-40]. Lysates were electrophoresed on 10–20% Tricine gels (Invitrogen, Carlsbad, CA) or 4–12% Bis-Tris NuPage gels (Invitrogen) and transferred to nitrocellulose. Western blotting was performed with anti-FLAG M2 (1:1000; Sigma, St. Louis, MO), anti-APP antibodies: C9 (1:1000, Selkoe laboratory), 22C11 (1:500, Millipore, Billerica, MA), anti-HA 3F10 (1:1000, Sigma), anti-myc A14 (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA); anti-C-terminal DISC1 (1:500, Invitrogen) and IRDye680- and IRDye800-conjugated secondary antibodies (1:10,000; Rockland Immunochemicals, Gilbertsville, PA), and detected using the LiCor detection system. Immunoprecipitations (Ips) were performed with anti-HA conjugated beads (Sigma) or with C9 and protein A and G (Sigma) and washed in lysis buffer. For endogenous co-IPs, adult rat brains were dounced in Tris buffered saline with protease inhibitors (Complete, Roche, Indianapolis, IN), run through a 27.5 gauge needle, and clarified by centrifugation at 1,000 g for 5 minutes. Supernatants were centrifuged at 100,000g for 1 hour to pellet membranes. Membrane pellet was lysed in 1% CHAPSO lysis buffer. Immunoprecipitations were performed from both the membrane and cytosolic fractions using C9 or C-terminal DISC1 antibody with a protein A and G mix.
Immunofluorescent staining and confocal microscopy
For analyses of in utero electroporations, paraformaldehyde-fixed brains were washed in PBS, embedded in 2% agarose, and vibratome sectioned (150 μm). Sections were incubated in blocking buffer (2% donkey or goat serum; 0.1% Triton X-100 in PBS) for >1 h. For BrdU immunostaining, sections were incubated in 2N HCl for 20 minutes at 37 degrees Celsius followed by incubation in sodium borate buffer (pH 8) and three PBS washes. Sections were then incubated in primary antibody (anti-MAP2, 1:10,000 (Abcam, Cambridge, UK); anti-Tbr1, 1:1000 (Abcam); anti-BrdU 1:100, (Accurate Biochemical (Axyll), Westbury, NY), anti-Ki67 1:50 (Dako, Carpinteria, CA) between 6 h and overnight at 4°C, followed by three washes in PBS. Sections were then incubated with Cy3 - and Cy5-conjugated secondary antibodies (1:500 –1:1000; Jackson Immunoresearch, West Grove, PA) for >2 h followed by four PBS washes. Sections were mounted on glass slides using GelMount (Biomeda, Foster City, CA). Images were acquired using a Zeiss (Oberkochen, Germany) LSM 510 confocal microscope with Axiovert 100M system.
For analyses of DISC1 localization following APP knockdown, primary cortical neurons were plated from Sprague Dawley rats from Charles River and cultured as described (Young-Pearse et al., 2008
). Neurons were plated onto LabTek CC2 coated two-well chamber slides (Thermofisher; Rochester, NY, USA) in 'plating media' (Dulbecco's modified Eagle's medium + 5% fetal calf serum + penicillin/streptomycin). 4 h after plating, media was changed to Neurobasal medium containing B27 supplement. 24 hours later, neurons were transfected with GFP alone, or with GFP and APP shRNA (Young-Pearse et al., 2007
), or else with GFP and a scrambled shRNA (Kamiya et al., 2005
) using Lipofectamine 2000 (Invitrogen). 48 hours later, neurons were fixed in 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), blocked in 2% donkey serum plus 0.1% Triton X-100, and immunostained using either anti-DISC1 (kindly provided by N. Brandon, (Brandon et al., 2004
)) and anti-gamma-tubulin (Abcam) or else with anti-DISC1 (Abcam) and anti-pericentrin (Abcam). Images were acquired using a Zeiss (Oberkochen, Germany) LSM 510 confocal microscope with Axiovert 100M system. GFP positive (transfected) neurons were categorized as having either a “focal” or “diffuse” pattern of DISC1 immunostaining. All analyses were performed blind to the condition being analyzed. Over 50 cells were analyzed per condition. Data from three independent experiments were acquired and analyzed. Significance was determined using Tukey-Kramer multiple comparisons tests.
Quantitative analyses of cortical plate entry
For quantitative analyses of migration, all electroporations were performed targeting the same region of the developing cortex. This resulted in a reliable electroporation of the dorsal-lateral region of the neocortex adjacent to the lateral ventricle. After harvest, brains were vibratome sectioned (100 μm) in the coronal plane and immunostained for microtubule-associated protein 2 (MAP2) to delineate the cortical plate. For each electroporation condition (i.e., each set of electroporated DNAs), greater than a total of 500 cells were counted and assessed for their location in either the MAP2+ cortical plate or the intermediate zone. To determine significant changes relative to control electroporations, at least three independent brains were electroporated and analyzed for each DNA condition. For each independent brain, the percentage of cells in the intermediate zone (IZ) and cortical plate (CP) were calculated. These values were then compared between electroporation conditions using GraphPad (San Diego, CA) InStat. Using this program, one-way ANOVA tests were performed with the Bonferroni multiple comparisons test. This analysis was used to determine that the percentage of cells in the CP in the noted electroporation conditions was significantly different from the percentage cells in the CP of control electroporations, or in the case of rescue experiments, of shRNA-receiving electroporations (similarly, these same conditions were statistically significant when comparing the percentage cells in the IZ for each condition).