UCH-L1 [LDN-57444 (LDN)] and UCH-L3 (4, 5, 6, 7-Tetrachloroindan-1,3-dione) inhibitors were purchased from Calbiochem (San Diego, CA). N-methyl D-aspartate (NMDA) and D(-)-2-amino-5-phosphonopentanoic acid (APV, NMDA receptor antagonist) were purchased from Tocris Bioscience (Bristol, United Kingdom). The HA-tagged Ubiquitin probe (HAUb-VME; vinyl methyl ester functionalized probe) was synthesized as described previously (Borodovsky et al., 2002
), and was provided by Dr. H. Ovaa (Netherlands' Cancer Institute, Netherlands).
UCH-L1 deficient mice
The UCH-L1 deficient and wild type litter mate mice (Uch-L1nm3419
) brains (8 weeks old) were obtained from Dr. Scott Wilson (University of Alabama, AL). This is a spontaneous mouse mutation that arose at Jackson Laboratory (Bar Harbor, ME) and subsequently mapped by Scott Wilson Group (Walters et al., 2008
The following antibodies were used in this study: mouse anti-Myc, rabbit anti-CDK5, rabbit anti-GFP, and rabbit anti-GKAP antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA); mouse anti-PSD-95 and rabbit anti-GluR1 were obtained from Calbiochem (San Diego, CA); rabbit anti-Shank antibody was a generous gift from Dr. Eunjoon Kim (KAISF, South Korea); rabbit anti-GluR1, mouse anti-NR1, and rabbit anti-NR2A antibodies were purchased from Upstate (Lake Placid, NY); rat anti-Homer and rabbit anti-Synapsin I (Chemicon, Bedford, MA); mouse anti-Bassoon (Stressgen, Ann Arbor, MI); chicken anti-Map2 (Abcam, Cambridge, MA); rabbit anti-UCH-L1 (Biomol, Plymouth Meeting, PA); rabbit anti-Ubiquitin (Dako, Carpinteria, CA); and rabbit anti-Vamp2 (Synaptic Systems, Goettingen, Germany).
Primary neuronal cultures
Hippocampal neuron cultures were prepared from P1 or P2 rat hippocampi as previously described (Patrick et al., 2003
). Briefly, for immunostaining experiments, rat hippocampi were dissected, dissociated by papain treatment and mechanical trituration, and plated at medium density (45,000 cells/cm2
) on poly-D-lysine coated coverslips (12 mm in diameter) or glass bottom dishes (MatTek, 35 mm, Ashland, MA). For biochemical experiments, mixed hippocampal and cortical neurons were plated at high density on 6 well plates (~500,000 cells per well) coated with poly-D-lysine. Cultures were maintained in B27 supplemented Neurobasal media (Invitrogen) until 14-21 days in vitro
Fractionations and DUB labeling assay
Fractions from rat brains were prepared as previously described in (Carlin et al., 1980
; Cho et al., 1992
). The DUB activity assay was done by incubating 20 μg of lysates from neuronal cultures or rat brain fractions with the HAUb-VME substrate in labeling buffer (50 mM Tris, pH 7.4, 5 mM MgCl2
, 250 mM sucrose, 1 mM DTT, and 1 mM ATP) for 1 h at 37°C. Proteins were then resolved on SDS-PAGE 4-20% gradient gels, and blots were subsequently probed with anti-HA monoclonal antibody. Labeled proteins were identified based on their migration on SDS-PAGE gels, and by comparison to previous published data where the specific bands were analyzed by mass spectroscopy (Borodovsky et al., 2002
Recombinant DNA and Sindbis constructs
The Sindbis EGFP viral construct was made by cloning the EGFP (Clontech, Mountain View, CA) open reading frame directly into pSinRep5 (Invitrogen, Carlsbad, CA). GFPu (in pEGFP-C1 plasmid backbone – Clontech, Palo Alto, CA), a fusion of the CL1 degron (degradation signal) on the carboxy terminus of GFP, was kindly provided by Ron Kopito (Stanford University, Palo Alto, CA). GFPu is ubiquitinated and specifically degraded by the ubiquitin proteasome system (UPS) (Gilon et al., 1998
; Bence et al., 2001
; Bence et al., 2005
). The Age
I fragment from photoactivatable (pa) GFP (a kind gift provided by Jennifer Lipponcott-Schwartz – National Institutes of Health, Bethesda, MD) was subcloned into the GFPu plasmid. paGFPu was then subcloned into pSinRep5 (Invitrogen, Carlsbad, CA). Orientation was verified by restriction digest and constructs were confirmed by DNA sequencing. The His6-Myc-Ubiquitin was provided by Dr. Ron Kopito (Stanford University, Palo Alto, CA) and was cloned into pSinRep5. The YFP-actin pSinRep5 plasmids was kindly provided by and Dr. E. Schuman (California Institute of Technology, Pasadena, CA). For production of recombinant Sindbis virions, RNA was transcribed using the SP6 mMessage mMachine Kit (Ambion, Austin, TX), and electroporated into BHK cells using a BTX ECM 600 at 220 V, 129 Ω, and 1050 μF. Virions were collected after 24-32 hours and stored at -80°C until use. For UCH-L1 expression constructs - the UCH-L1 open reading frame was obtained from Incyte full length human cDNA clone (Open Biosystems, Huntsville, AL) encoding wild type UCH-L1 and was amplified by PCR with a 5′-oligo containing an Xho
I site and a 3′-oligo containing an Age
I site, and subsequently cloned in the pEGFP-N1 vector. The single point mutations in the UCH-L1 DNA were introduced by PCR-based site-directed mutagenesis of template plasmid cDNA using primers designed to introduce specific mutations (C90S, 5′-CCATTGGGAATTCCTCTGGCATCGGAC-3′, and D30A, 5′-TTCGTGGCCCTGGGGCTG-3′). All constructs were verified by sequencing and by expression of proteins of the expected molecular weight in HEK 293T cells.
Drug treatments and virus infections
For protein expression analysis by Western blotting or immunofluorescence staining experiments, cultured neurons were treated with 10 μM of UCH-L1 (LDN) or UCH-L3 inhibitor for 24 hr. In experiments where neurons were subjected to LDN treatment and infections, neurons were first treated with LDN and then infected by adding virions directly to the culture medium and allowing protein expression for 12-14 hr. The total time of exposure to LDN was kept constant (24 hours). Activity stimulation experiments were performed by treating cultures with NMDA and glycine at 50 and 10 μM, respectively, for 10 min at 37°C. Where indicated, neurons were pre-treated with UCH-L1 inhibitor (10 μM) for 24 hr or APV (50 μM) for 10 min before addition of NMDA/glycine to the culture media.
At the end of each experiment, hippocampal neurons plated on coverslips or 35 mm glass bottom dishes were rinsed briefly in PBS and fixed with 4% paraformaldehyde (PFA) and 4% sucrose in PBS-MC (phosphate buffered saline with 1 mM MgCl2 and 0.1 CaCl2) for 10 min at room temperature. Neurons were then rinsed 3× with PBS-MC and subsequently blocked and permeabilized with blocking buffer containing (2% BSA, 0.2% Triton* X-100 in PBS-MC) for 20 min. After rinsing neurons 3× with PBS-MC, primary antibodies were added in blocking buffer and cultures were incubated overnight at 4°C. The following antibodies and dilutions were used for immonofluorescence stainings: mouse anti-PSD-95 (1:500), rabbit anti-Synapsin I (1:2000), mouse anti-Bassoon (1:2000), rabbit anti-Shank (1:2000), rabbit anti-GluR1 (1:20), chicken anti-Map2 (1:5000), mouse anti-Myc (1:1000). After three washes with PBS-MC, neurons were incubated in goat anti-rabbit, -mouse or -chicken secondary antibodies conjugated to Alexa 488, Alexa 568, or Alexa 678 (1:500 each; Molecular Probes) at room temperature for 1hr. Neurons were washed 3× with PBS-MC and mounted on slides with Aqua Poly/Mount (Polysciences, Warrington, PA). For live-labeling of surface GluR1, the anti-GluR1 antibody against the N-terminus extracellular epitope of the receptor was added to neurons in culture medium at 1:20 dilution for 10 min before washing out excess antibody and fixing with 4% PFA/4% sucrose.
Mature hippocampal neurons (>21 DIV) were plated in 35 mm glass bottom dishes and treated with DMSO (control) or LDN (10 μM). After 24 h, cells were fixed in 2% paraformaldehyde and 1% glutaraldehyde, then fixed in osmium tetraoxide and embedded in epon araldite. Once the resin hardened, blocks with the cells were detached from each dish and mounted for sectioning with an ultramicrotome (Leica). Grids were stained with 1% uranyl acetate and analyzed with a Zeiss OM 10 electron microscope as previously described (Rockenstein et al., 2001
). Manual analysis of PST diameter, vesicle number, and synaptic contact zone was performed. A total of 10 micrographs were obtained and from each grid (9 grids per condition) for a total of 90 electron micrographs analyzed per condition. Analysis was performed using ImageQuant. Magnification = 30,000×. Statistical significance was determined by unpaired two-tailed Student's t test.
Live Imaging of paGFPu Degradation
Cultured hippocampal neurons (>21 DIV) on 35 mm glass bottom dishes were incubated for 24 hr prior to imaging in media containing either DMSO (control) or LDN (10 μM). paGFPu virions were added directly to culture media after 12 hr of LDN treatment and protein expression was allowed to continue for 12-14 hours. Culture media was then replaced with warm HBS (HEPES buffered saline solution containing in mM: 119 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 30 Glucose, 10 HEPES). Cells were maintained at ~35°C using a ceramic heat lamp (ZooMed, San Luis Obispo, CA) and bath temperature was continually monitored by a digital probe thermometer. Infected neurons (identified by mCherry expression) were then photoactivated for 10-15 seconds with 100 W Hg2+ lamp and a D405/40× with 440 DCLP dichroic filter set (Chroma). For live imaging, pyramidal-like neurons were selected in a random fashion. Confocal Z-stack images (with 0.5 μm sections) were acquired with a 63× objective every 2 minutes.
In Vitro Proteasome Activity Assay
Proteasome activity was measured as previously described with slight modifications (Kisselev and Goldberg, 2005
). Briefly, cultured neurons were incubated for 24 hr in media containing either DMSO (control) or LDN (10 μM). Neurons were then lysed in proteasome assay buffer (50 mM Tris-HCl, pH 7.5, 250 mM Sucrose, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP, 1 mM DTT and 0.025 % Digitonin) for 15 min on ice. Lysates were cleared by centrifugation at 14,000 rpm for 15 min. 100 μM of the fluorogenic proteasome peptide substrate Suc-LLVY-AMC (Biomol) was then added to equal amounts of cleared lysates in a 96-well microtiter plate. Fluorescence (380 nm excitation, 460 nm emission) was monitored on a microplate fluorometer (HTS 7000 Plus, Perkin Elmer, Boston, MA) every 5 min for 2 hr at room temperature.
Western Blot Analysis
Cultured neurons were lysed in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton* X-100, 0.1% SDS, 1 mM EDTA) containing protease inhibitors (Roche, Indianapolis, IN). Rat or mouse brains were homogenized in RIPA buffer at 900 rpm in teflon-glass homogenizers. Neuronal cell lysates or brain homogenates were centrifuged at 14,000 rpm and supernatants were removed and protein concentration was determined by BSA™ Protein Assay Kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Protein samples were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were then blocked for 1 hr in TBST blocking buffer (TBS, 0.1% Tween 20, and 5% milk) at room temperature and then incubated with primary antibodies in blocking buffer overnight at 4°C. The antibodies used were at the following dilutions: mouse anti-PSD-95 (1:5000), rabbit anti-Synapsin I (1:10,000), rabbit anti-GluR1 (1:5000), rabbit anti-Shank (1:10,000), rabbit anti-GFP (1:10,000), rabbit anti-UCH-L1 (1:5000), rabbit anti-GKAP (1:2000), rat anti-Homer (1:2000), mouse anti-NR1 (1:2000), rabbit anti-NR2A (1:2000), rabbit anti-Ubiquitin (1:2000), rabbit anti-Vamp2 (1:5000), rabbit anti-CDK5 (1:10,000). Blots were then washed 3× in TBST washing buffer (TBS, 0.1% Tween 20) and incubated with goat anti-rabbit, -mouse or -rat IgG conjugated to horseradish peroxidase (1:5000). Protein bands were visualized by Chemiluminescence plus reagent (PerkinElmer) and were digitized and quantified using NIH ImageJ software. For statistical analysis unpaired Student t-test was performed between any two conditions.
Image acquisition and quantification
Confocal images were acquired using a Leica (Wetzlar, Germany) DMI6000 inverted microscope outfitted with a Yokogawa (Tokyo, Japan) Spinning disk confocal head, a Orca ER High Resolution B&W Cooled CCD camera (6.45 μm/pixel at 1×) (Hamamatsu, Sewickley, PA), Plan Apochromat 40×/1.25 na and 63×/1.4 na objective, and a Melles Griot (Carlsbad, CA) Argon/Krypton 100 mW air-cooled laser for 488/568/647 nm excitations. Exposure times were held constant during acquisition of all images for each experiment. Pyramidal-like cells were chosen in a random fashion. Confocal z-stacks were taken at 0.4-0.5 μm depth intervals in all experiments. For image analysis, max Z-projections were utilized. Images were thresholded equally 1.5-2 times above background. Dendrites from individual neurons were then straightened and used for analysis. Fluorescence intensity associated with pre- and postsynaptic protein puncta was measured to determine the size and number of puncta (normalized to dendritic length) in control and LDN-treated neurons. GFP-expressing hippocampal neurons were used for spine morphology analysis using the Edgefitter NIH ImageJ plugin (Ghosh Lab, UCSD). To determine the length of a spine, the distance from the protrusion's tip to the dendritic shaft was measured manually. To measure the width of a spine, the maximal length of the spine head perpendicular to the long axis of the spine neck was measured. The number of spines visible along the dendrite was counted manually per 1 μM dendritic length. Measurements were then automatically logged from NIH ImageJ into Microsoft Excel. Statistical significance was determined by unpaired two-tailed Student's t test. All imaging and analysis of ubiquitin rescue experiments () were performed in a blinded fashion. For quantification of the proteasome reporter degradation, images were thresholded above background equally between conditions. Total integrated fluorescence intensity was measured from dendrites at each time interval and expressed as the percent change from time zero. Grouped analysis of dendritic fluorescence decay over time from each treatment group was plotted as line graphs (mean ± SEM). The degradation rate of reporter fluorescence decay was obtained by taking the difference of total fluorescence loss (arbitrary units = AU) over time (Fi − Fn/timen) from individual experiments. The mean rate ± SEM per treated group was then normalized to the control rates. For statistical analysis, grouped degradation rates were analyzed by unpaired two-tailed t-tests. Half of the paGFPu reporter degradation experiments were performed in a blinded fashion. There was no significant difference between experiments performed blind and unblind experiments and therefore the data was combined.
Overexpression of ubiquitin restores synaptic structure in LDN-treated neurons