Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) delays Alzheimer’s progression in vivo
Mingming Zhang 1, Fang Cai 1, Shuting Zhang 1, Si Zhang 1, and Weihong Song 1,2
1 Townsend Family Laboratories, Department of Psychiatry, Brain Research Center, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada.
2 To whom correspondence should be addressed: Dr. Weihong Song, MD, PhD, FCAHS, Department of Psychiatry, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada; Tel: 604-822-8019; Fax: 604-822-7756. Email:
Abbreviations:Alzheimer’s disease, AD; amyloid β precursor proteins, APP; Beta-site APP cleaving enzyme 1, BACE1; Amyloid β protein, Aβ.
Key Words: APP; Aβ; UCHL1; Alzheimer’s Disease
Online Supplemental Material and Methods
Reagents. Dulbecco’s modified Eagle’s medium was purchased from Invitrogen Life Technologies. Anti-β-actin mAb AC-15 was obtained from Sigma-Aldrich. Anti-UCHL1 mouse mAb BH7 was purchased from Novus Biologicals. MG132, chloroquine, UCHL1 inhibitor LDN-57444 and anti-ubiquitin rabbit pAb (662099) were obtained from Calbiochem. Anti-BACE1 rabbit mAb D10E5 was from Cell Signaling Technologies. Rabbit C20 that recognized last 20 C-terminal amino acids of APP was generated previously by our laboratory 1. IRDyeTM680-labelled goat anti-rabbit and IRDyeTM800CW-labelled goat anti-mouse antibodies were from LI-COR Biosciences.
cDNA constructs. To generate the adeno-associated virus (AAV) expressing UCHL1, the human UCHL1 cDNA sequence was cloned into the pAAV-GFP-cDNA6 vector (Vector Biolabs). The pAAV-GFP-cDNA6 vector was used as the control plasmid. Separate CMV promoters allowed the bi-cistronic expression of the inserted transgene with enhanced GFP (eGFP), resulting in UCHL1 and GFP expression as individual proteins within transduced cells. The recombinant AAV1-UCHL1-GFP and AAV1-GFP were made by combining AAV2-based genomic constructs with cap genes derived from AAV serotype 1 (Vector Biolabs).
Cell cultures, transfection and pharmacological treatment. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% sodium pyruvate, 1%l-glutamine and 1% penicillin/streptomycin (Invitrogen). HAW cells, the HEK293 cell line that stably overexpresses wildtype APP, and 20E2 cells, the HEK293 cell line that stably overexpresses Swedish mutant APP695, were cultured in complete DMEM with 50 μg/ml geneticin. All cells were maintained at 37°C in an incubator containing 5% CO2. Transient transfections of plasmids were performed using the calcium phosphate method or Lipofectamine 2000. LDN-57444 (10 μM) was applied to cells to specifically inhibit UCHL1 hydrolase activity. Chloroquine (100 μM) was used to inhibit lysosomal protein degradation. MG132 (5 μM) was used to inhibit proteasomal protein degradation 2.
Animal study. Animal experiment protocols were approved by The University of British Columbia Animal Care and Use committee.APP23 transgenic mice carry human APP751 cDNA with the Swedish mutation at positions 670/671 (KM/NL) under control of the murine Thy-1.2 promoter 3,4. PS45 transgenic mice carry human PS1 cDNA with the G384A mutation identified in a family with early-onset FAD under control of the murine Thy-1 promoter 5,6. The gracile axonal dystrophy (gad) mouse carries a spontaneous mutation with an in-frame deletion in exons 7 and 8 ofUCHL17, and is equivalent to a UCHL1 knockout mouse model. Heterozygousgadmice were originally generated and kindly provided by Dr. Keiji Wada at the National Institute of Neuroscience of Japan.The APP23/PS45 double transgenic mice were generated by breeding the APP23 hemizygous strain with the PS45 homozygous strain. The APP23/gad mice were generated by breeding the APP23 hemizygous strain with the gad heterozygous strain. The genotypes of the mice were confirmed by PCR from DNA extracted from ear tissue. All animal experiments were approved and conducted in accordance with the University of British Columbia Animal Care and Use Committee guidelines.
Virus injection. Seven-week-old APP23/PS45 mice were anesthetized by isoflurane (1-2%) and secured on the stereotaxic frame (Kopf Instruments). Meloxicam (2mg/kg) was injected subcutaneously before the skull was exposed.A small section of the skull was removed using a micro drill (Fine Science Tools, 0.5mm tip diameter) at the co-ordinates of 2.0 mm rostral and 2.0 mm lateral to the bregma. One microliter of AAV1-UCHL1-GFP or AAV1-GFP (1013GC/ml) was injected bilaterally or unilaterally (1.8 mm ventral from the dura) at a rate of 0.2 µl/min. After each injection, the needle was left in place for an additional 2 minutes before withdrawal.
Morris water maze test. The Morris Water Maze test was performed as previously described (Qing et al., 2008)8. Briefly, the test was performed in a 1.5-meter diameter pool with a 10-cm diameter platform placed in the southwest quadrant of the pool. The procedure consisted of one day of visible platform tests and 4 days of hidden platform tests, plus a probe trial 24 h after the last hidden platform test. In the visible platform test, the platform was placed 1 cm above the clear water surface. The position of platform and the starting direction varied across trials. Mice were tested for 5 continuous trials with an inter-trial interval of 75 min. Mice were allowed to swim for 60 sec if they could not find the platform. Failed mice were gently guided to the platform and remained there for 20 sec before they were sent back to their home cages. In the hidden platform tests, the platform was submerged 1 cm below an opaque water surface in a fixed position in the southwest/third quadrant. In the probe trial, the platform was removed and mice were allowed to swim for 60 sec. Mouse behavior including speed and escape latency was automatically video-recorded by automated video tracking (ANY-maze, Stoelting).
Immunohistochemistry, immunoblotting and immunoprecipitation. The immunohistochemical staining was performed as previously described 9,10. Briefly, mice were sacrificed and half of the brains were fixed in 4% paraformaldehyde, followed by 30% sucrose solution, and sectioned with a Leica Cryostat to 30 μm thickness after embedding in O.C.T. solution. Every 12th slice with the same reference position was mounted onto the slides for staining. The slices were stained with biotinylated monoclonal 4G8 antibody. Plaques were visualized by the ABC and DAB method and photos were taken under microscopy at 40X magnification. Plaques were quantified and the average plaque counts per slice were recorded for each mouse. Thioflavin-S staining of plaques was performed with 1% thioflavin-S and the green fluorescence stained plaques were visualized using fluorescence microscopy. For GFP detection in the hippocampus, GFP-expressing cells were visualized by microscopy. For exogenous UCHL1 detection, brain slices were stained by anti-UCHL1 antibody BH7, and visualized by goat anti-mouse Alexa594 dye (Life Technologies). For immunoblotting,cells were lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS and protease inhibitor cocktail Complete, pH 7.2) (Roche Molecular Biochemicals). Brain tissues were lysed in RIPA buffer with 1% SDS. Cell lysates were briefly sonicated and centrifuged at 14,000 rpm for 10 min. The samples were diluted in 4X SDS-sample buffer and loaded onto 8% tris-glycine, 12% tris-glycine or 16% Tris-Tricine SDS-PAGE and transferred to PVDF-FL membranes. Membranes were blocked for 1 h in PBS containing 5% non-fat dried milk followed by overnight incubation at 4℃ in primary antibodies diluted in the blocking medium. UCHL1 was detected using the anti-UCHL1 antibody BH7 (Novus Biologicals). β-actin was detected using monoclonal antibody AC-15 (Sigma). The membranes were incubated with IRDye 800CW-labelled goat anti-mouse or IRDye 680CW-labelled goat anti-rabbit antibodies in PBS with 0.1% Tween-20 at 22℃ for 1 h, and visualized on the Odyssey system (LI-COR Biosciences). To perform immunoprecipitation assays, cells were lysed in NP-40 buffer (10 mM Hepes, pH 7.5, 142.4 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% NP-40, and Roche protease inhibitor cocktail Complete), sonicated and centrifuged at 14,000g for 20 min. The supernatant was pre-cleared with Sepharose CL-4B for 1 h. Primary capturing antibodies were incubated with protein A/G sepharose beads (Santa Cruz) for 1 h before incubation with the protein lysates at 4℃ overnight. The following day the immunoprecipitates were washed three times with NP-40 buffer and one time with PBS on ice. Sample loading buffer was added to the pellets and samples were boiled for 5 min before they were subjected to western blot analysis. Co-immunoprecipitation was performed using Pierce Crosslink IP Kit (MF157118). Briefly, primary capturing antibodies were incubated with Pierce Protein A/G Plus Agarose at room temperature for 1 h in Coupling Buffer. The bound antibodies were then crosslinked with Pierce Protein A/G Plus Agarose by DSS for 1h at room temperature to prevent antibodies from co-eluting with the antigens later. Haw cells or 20E2 cells that had undergone transfections or treatments were harvested and lysed in IP Lysis/Wash Buffer (0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP-40, 5% glycerol; pH 7.4). Lysates were pre-cleared by Control Agarose Resin slurry at 4℃ for 1 h and added to the antibody-crosslinked resin for incubation overnight at 4℃. The following day samples were washed and eluted using Elusion Buffer. 5X Lane Marker Sample Buffer with 100mM DTT were added to samples to make a 1X final solution. Samples were heated at 95-100°C for 5 min before western blot analysis.
Aβ ELISA. Tissue extracts from transgenic mouse hippocampal regions were collected and prepared according to manufacturer’s instructions. Briefly, tissues were homogenized in 5 M guanidineHCl/50 mM Tris HCl (pH 8.0) and allowed to mix at room temperature for 4 h. The samples were then diluted in ice-cold reaction buffer (PBS with 5% BSA, 0.03% Tween-20, and supplemented with AEBSF and Roche mini protease inhibitor cocktail tablet). The final guanidine HCl concentration was less than 0.1M. The concentration of Aβ40 was detected using Aβ40 Human ELISA Kit (Invitrogen).
Statistics.All results were presented as mean ± SEM and analyzed by ANOVA or 2-tailed Student’st-test. One-way ANOVA was followed by post-hocNewman-Keuls test. Two-way ANOVA was followed by post-hocBonferroni test. Statistical significance is accepted whenp< 0.05.
Online Supplemental Figures
Supplementary figure 1
Supplemental Figure 1. rAAV-mediated transgene expression of UCHL1 in the hippocampal region of APP23/PS45 mice. (A)APP23/PS45 mice at the age of seven weeks old were intracranially injected with AAV1-UCHL1-GFP or AAV1-GFP control into the hippocampal region. One microliter of rAAV (1013GC/ml) was injected bilaterally (2.0 mm rostral to bregma, 2.0 mm lateral to bregma, 1.8 mm ventral from the dura) 11 at a rate of 0.2 µl/min. Eight weeks post-injection the mice underwent behavioral tests before they were sacrificed. (B) Hippocampal tissues from saline-injected APP23/PS45 mice and mice that underwent treatment described in (A) were lysed in RIPA buffer for protein detection. EGFP was detected by rabbit anti-GFP antibody made in-house. UCHL1 was detected by anti-UCHL1 antibody BH7 (Novus). EGFP was detected in both AAV1-GFP- and AAV1-UCHL1-GFP-injected hippocampi but not in saline-injected hippocampus. UCHL1 level was increased in AAV1-UCHL1-GFP-injected hippocampi compared to those treated with AAV1-GFP.β-actin served as protein loading control. (C) Mice were unilaterally injected with AAV1-UCHL1-GFP or AAV1-GFP to the left hippocampi at 7 weeks of age and were sacrificed 10weeks post-injection. EGFP was robustly expressed in the left hippocampi of both mice whereas only trace amount was detected in the right hippocampi. UCHL1 was increased in the left hippocampus of AAV1-UCHL1-GFP-injected mouse but not AAV1-GFP-injected mouse. (D) The brains of mice that were unilaterally injected with AAV1-UCHL1-GFP to the left hippocampi were sectioned to 30 μm thickness. EGFP-expressing cells were visualized by microscopy. EGFP was robustly expressed in the dentate gyrus and CA3 area of the left hippocampus (a), whereas only background was observed in saline-injected right hippocampus (b). (E) Hippocampi of mice from (A) were stained with anti-UCHL1 antibody. EGFP and UCHL1 expression were visualized by microscopy. CA3 neurons in both AAV1-GFP- and AAV1-UCHL1-GFP-injected mice were positive with eGFP expression (a and b). UCHL1 was co-overexpressed with eGFP in CA3 neurons of AAV1-UCHL1-GFP mice (d), whereas no UCHL1 was detected in AAV1-GFP mice (c).
Online Supplemental References
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