Generation of LRRK2 G2019S conditional transgenic mice
To develop a conditional LRRK2 G2019S transgenic mouse model, a cDNA fragment encoding the C-terminal hemagglutinin (HA)-tagged G2019S mutant LRRK2 protein was inserted into the mouse prior protein (pPrP)—tetP gene expression vector (a gift from Dr. David Borchelt, University of Florida, Gainesville, FL), which is controlled by the tetracycline-responsive promoter (tetP) (Jankowsky et al., 2005
). The LRRK2 expression construct was then purified and microinjected into fertilized oocytes derived from C57BL6/J mice. The founder mice were crossed with wild-type (WT) C57BL6/J mice to produce the F1 generation. The F1 LRRK2 G2019S mutant mice were mated with calcium/calmodulin-dependent kinase II (CaMKII)—tTA mice (Mayford et al., 1996
) to achieve high expression of LRRK2 in forebrain regions, including the olfactory bulb, striatum, hippocampus, and cortex. With the tet-off system, the expression of human LRRK2 was almost completely (>90%) suppressed after feeding the mouse with doxycycline (Dox)-treated pellets for 4 weeks (data not shown).
Mouse forebrains were dissected out and homogenized in sucrose buffer [0.32 M sucrose, 1 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2, and protease inhibitor mixture (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Pierce, Rockford, IL)]. Protein extracts (1 mg) were used for immunoprecipitation (IP), which were diluted in 1000 μl of cold IP buffer (20 mM HEPES, pH7.5, 1% NP-40, 5% glycol, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, protease inhibitor mixture, and phosphatase inhibitor cocktail) and then incubated with 20 μl of HA matrix (anti-HA antibody-conjugated agarose beads; Roche) for 3 h at 4°C. The beads were washed with cold IP buffer six times and then eluted with 20 μl of triethylamine buffer (100 mM, pH 11). The eluted samples were neutralized by 5 μl of Tris-HCl (1 M), pH 7.4, and then size fractioned by SDS-PAGE. For immunoprecipitation from human embryonic kidney-293 (HEK-293) cells, cells were homogenized in cold IP buffer, and 500 μg of protein was used for immunoprecipitation using HA matrix as described previously. The beads were boiled in the SDS-PAGE sample buffer for 5 min, and the supernatants were size fractioned by SDS-PAGE.
Silver staining and mass spectrometry
The LRRK2-binding proteins isolated by HA affinity purification was visualized by silver staining using the Silver Quest Silver Staining kit (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. Visible protein bands were excised from the silver-stained gel and cut into 1 mm pieces. Proteins were identified by mass spectrometry performed by the Taplin Biological Mass Spectrometry Facility at Harvard Medical School (Boston, MA). A total of 14 Hsp90α-related peptides and 5 Hsp90β-related peptides were identified.
Neurons and HEK-293 cells were lysed in 20 mM HEPES, pH 7.4, 0.5% Triton X-100, 2% SDS, protease inhibitor mixture, and phosphatase inhibitor cocktail. Protein was size fractioned by 4–12% NuPage BisTris-PAGE (Invitrogen) using 3-(N-morpholino)-propanesulfonic acid running buffer (Invitrogen) and transferred to polyvinylidene difluoride membranes. Antibodies specific for LRRK2 (1:1000, JH5514), Hsp90 (1:3000; BD Biosciences Transduction Laboratories, Lexington, KY), cell division cycle 37 (Cdc37) (1:1000; BD Biosciences Transduction Laboratories), Hsp70 (1:3000; BD Biosciences Transduction Laboratories), HA (1:1000; Roche), α-synuclein (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (1:3000; Sigma) were used in this study. The enhanced chemiluminescence method was used to visualize the bound antibodies.
Cell culture and treatment
Procedures for primary cultured cortical and hippocampal neuron cultures from postnatal day 1 pups and for HEK-293 cells were as described previously (Cai et al., 2005
; Lai et al., 2006
; Wang et al., 2008
). When indicated, proteasome, lysosome, and protein synthesis inhibitors were preincubated with cells before treatment of Hsp90 inhibitors and kept in the culture medium. Geldanamycin (GA) (Sigma) and MG132 (Sigma) were dissolved in DMSO. Chloroquine (Sigma) was dissolved in water. PU-H71 and PU-DZ8 were dissolved in PBS buffer.
LRRK2 expression constructs and transfection
The C-terminal HA-tagged wild-type and G2019S mutant LRRK2 cDNA were cloned into the pPrP—tetP tetracycline-regulated gene expression vector as indicated above. The cytomegalovirus (pCMV)—reverse tetracycline transactivator (rtTA) vector is a gift from Dr. Hermann Bujard (University of Heidelberg, Heidelberg, Germany). FuGene6 reagent (Roche) was used for cell transfection as suggested by the manufacturer. Dox (Sigma) was added into culture medium at the final concentration of 250 ng/L to turn on the LRRK2 expression in pPrP—tetP—LRRK2 and pCMV—rtTA cotransfected cells. In the PU-H71 dose-dependent study, HEK-293 cells were transfected with constructs for 24 h and then treated with different concentration of PU-H71 for 24 h. To disrupt the LRRK2—Hsp90 complex, culture cells were exposed to 1 μM GA for 4 h and then homogenized for immunoprecipitation.
HaloTag Interchangeable Labeling technology (Promega, Madison, WI) was used to study the stability of LRRK2 protein. Wild-type and G2019S mutant LRRK2 cDNA were cloned into the HaloTag pHT2 vector. After transfection with HaloTag wild-type or G2019S mutant LRRK2 vector for 36 h, HEK-293 cells were pulse-labeled with 5 μM HaloTag TMR Biotin ligand (Promega) in DMEM plus 10% FBS for 3 h. After washing three times with PBS, cells were chased in DMEM plus 10% FBS in the presence or absence of 1 μM PU-H71 for the indicated time. Cell were lysed in 20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, and protease inhibitor cocktail (Roche). The biotin-labeled LRRK2 was captured by streptavidin-coated particles and Magnesphere Paramagnetic particles (Promega) for 2 h at 4°C and then washed three times with PBS containing 150 mM NaCl and 1% Triton X-100 and once with PBS only. Biotinylated LRRK2 was eluted by heating in SDS-PAGE sample buffer for 5 min at 95°C. Proteins were resolved on SDS-PAGE gel and detected by immunoblot using Halo antibody (Promega).
RNA extraction and reverse transcriptase-PCR
Total RNA was extracted using RNAeasy Mini kit according to the instructions of the manufacturer (Qiagen, Valencia, CA). Reverse transcriptase (RT)-PCR was performed using SuperScript III first-strand synthesis system for RTPCR (Invitrogen). RNase-treated cDNA was used for real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7700 Sequence Detection system. The cDNA of β-actin was used as an internal control. The following primers were used for LRRK2 (5′-AGCAGGACAAAGCCAGCCTCA-3′ and 5′-GATGGCAGCATTGGGATACAG-3′) and β-actin (5′-CGTTGACATCCGTAAAGACC-3′ and 5′-GCTAGGAGCCAGAGCAGTAA-3′).
Quantification of axonal outgrowth
Hippocampal neurons were fixed after 2 d in culture and probed with TuJ1, a monoclonal neuron-specific antibody against βIII-tubulin. The images were captured using a Zeiss (Thornwood, NY) confocal microscope (LSM 510 META), and the lengths of axons were measured by using the segmented line function of NIH ImageJ software. In each experimental group, the lengths of axons were represented as a mean of axons from 50–100 neurons sampled from two to three randomly selected microscopic fields of three to four independent cultures.
Data on drug-dose dependency and LRRK2 half-life (t1/2) were analyzed by nonlinear regression analysis using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). The IC50 values were expressed as mean ± 95% confidence intervals (CIs). Statistical significances were determined by comparing datasets of different groups using F test of the best-fit values for three parameters (logIC50, top and bottom). Statistical analysis of axon length was performed using the Stat-View program (version 5.0; SAS Institute, Cary, NC). Data are presented as means ± SEM. Statistical significances were determined by comparing means of different groups using ANOVA, followed by Fisher’s PLSD test.