Polyclonal antibodies against profilin-1 and -2a were generated by Open Biosystems (Huntsville, AL) by injecting into rabbits the linear peptides KCYEMASHLRRSQY and KAYSMAKYLRDSGF conjugated to KLH. Following immunization, blood was collected, and crude serum was purified with an affinity column containing the respective profilin peptide. Other antibodies used include a monoclonal anti-chickadee, chi1J (Developmental Studies Hybridoma Bank, University of Iowa), monoclonal anti-actin (Chemicon), monoclonal anti-tubulin (Chemicon), monoclonal anti-actin (AC-40; Sigma-Aldrich), monoclonal anti-GFP (BD Biosciences), and rabbit anti-profilin (Alexis Biochemicals).
Frozen blocks of human cortical tissue from Brodmann area 9 were obtained for western blotting from the Harvard Brain Tissue Resource Center and Vanderbilt University Medical Center in accordance with institutional guidelines (NIH, Office of Human Subjects Research). Specimens were matched for postmortem interval (PMI) and divided into groups based on supplied information: (a) controls (n = 3, age = 65 ± 16 years, PMI = 10 ± 2 hours); (b) presymptomatic HD gene carriers (n = 3, age = 46 ± 31 years, PMI = 13 ± 6 hours); (c) grade 1 HD patients (n = 3, age = 60 ± 10 years, PMI = 13 ± 7 hours); (d), grade 4 HD patients (n = 3, age = 67 ± 6 years, PMI = 16 ± 7 hours). Neuropathological classification was based on previously established guidelines (Vonsattel et al., 1985
). The mutant alleles in the HD patients ranged from 45 to 50 CAGs.
The cell model used for these experiments, a stably transfected, inducible PC12 cell line expressing exon 1 of the HD gene with either 23 or 74 CAGs under the control of a tetracycline promoter, has been previously described (PC12–23Q and PC12–74Q, (Wyttenbach et al., 2001
)). The cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) with 100 U/ml penicillin/streptamycin, 2 mM L-glutamine, 10% horse serum (HS), 5% Tet-free fetal bovine serum (FBS), and 100 µg/ml G418 at 37° C and 10% CO2
. Induction of htt expression was done by incubating the cells in media containing 1 µg/ml of doxycycline for the specified periods of time. For differentiation, the cells were incubated with 100 ng/ml of nerve growth factor (NGF) in media containing 1% HS and no FBS.
The htt-inducible PC12 cells were incubated in media containing 1 µg/ml doxycycline for 48 hr. For the specified experiments, cells were transfected with empty vector (mock) or with profilin-1 cDNA using Fugene HD as per manufacturer’s instructions (Roche). Following induction cells were scraped, washed in phosphate-buffered saline (PBS), and then lysed in 0.75 ml of actin lysis buffer (50 mM NaCl, 5 mM MgCl2, 1 mM ATP, 5% glycerol, 5 mM EGTA, 0.1% Triton X-100, 0.1% Nonidet P-40, 0.1% Tween 20, 0.1% β-mercaptoethanol, 50 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9). The F-actin and G-actin pools were separated by ultracentrifugation at 100,000 × g at 30 °C. The supernatant was diluted 1:2 with Laemmli buffer, and the pellet was resuspended in cold distilled H2O with 1 µM cytochalasin D and sonicated for 10 s. The pellet fraction was kept on ice for 45 min and then diluted 1:4 with Laemmli buffer. Both fractions were then boiled for 5 min and centrifuged at 14,000 × g for 10 min at 4 °C to remove remaining connective tissue. The same relative amounts of supernatant and pellet fractions (2:1) were loaded on 10% polyacrylamide gels and analyzed by Western blot using an anti-actin antibody (AC-40; Sigma-Aldrich). The F/G-actin ratio was determined by scanning densitometry. The values are presented as the means ± standard error. Statistical comparisons were done using Student’s t-test (p values < 0.05 were considered statistically significant).
The htt-inducible PC12 cells were seeded in 35 mm dishes at 4 × 105 cells / well. On day 1, the cells were transfected with empty vector (mock) or with profilin-1 cDNA using Fugene HD as per manufacturer’s instructions (Roche). The following day, cells were induced with 1 µg/ml doxycycline. 72 h following induction, the cells were trypsinized and centrifuged at 800 × g for 5 min at room temperature. The cell pellets were washed and re-suspended in 400 ul PBS. Propidium iodide (PI) was added to the cell suspension at 1 µg/ml. A total of 50,000 cells were collected and analyzed by flow cytometry (Becton Dickinson FACS Calibur). PI positive cells were detected using a 530 nm excitation and 617 nm emission filter. Cell populations were gated on size (forward scatter) and granularity (side scatter) to exclude debris and cell clumps. An uninduced, unstained negative control sample and a positive control sample stained with PI were used to establish reference regions. Cell death was calculated as the percent cells that were PI positive and reported as the mean ± standard error of three independent experiments. Statistical comparisons were performed using Student’s t-test (P values < 0.05 were considered statistically significant).
For human specimens, pieces of frozen cortical tissue were immersed in liquid nitrogen and pulverized with a BioPulverizer™ (Biospec Products, Bartlesville, OK). Powdered tissue was homogenized in a buffer consisting of 25 mM Tris-HCl (pH 7.5), 5 mM EDTA, 1% SDS, and protease inhibitors (Complete Mini, Roche) at 1:10 w/v. The samples were sonicated on ice for 10 seconds three times and then centrifuged at 14,000 g for 10 minutes at 4° C to remove any particulate matter. For cells, at specified time points, the samples were washed with PBS, scraped, collected, and lysed in ice-cold RIPA buffer (50 mM Tris-HCl, 1% NP40, 0.5% Na-DOC, 150 mM NaCl, 0.1% SDS, and 2 mM EDTA) containing protease inhibitors. The cell lysate was sonicated on ice and incubated for 20 minutes on ice. For Drosophila, flies were frozen on dry ice and the heads were collected on a frozen plate and transferred to an eppendorf tube with RIPA buffer (1:5 w/v). The heads were then homogenized with a pestle and sonicated on ice. The lysates were centrifuged at 15,000 g for 20 minutes at 4° C for 20 minutes. Afterwards, for all samples the supernatant were collected and kept on ice. Protein concentrations for all samples were determined using the Pierce BCA protein assay (Rockford, IL). Laemmli buffer (BioRad, Hercules, CA) was added to each supernatant, and the samples were heated to 95° C for 5 minutes. Equal protein amounts of cell extract (20 or 60 µg total protein) were separated by SDS-PAGE and then transferred to nitrocellulose. The blots were then incubated in 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) for 1 hour at room temperature. The blocking solution was then removed, and each blot was incubated overnight at 4° C in a solution of primary antibody diluted as indicated in PBS-T (anti-profilin, 1:1000; anti-profilin-1, 1:1000; anti-profilin-2a 1:1000; anti-chickadee 1:10; anti-actin 1:5000; anti-tubulin 1:3000). The blots were then washed three times in PBS-T and incubated with either anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (Amersham, Buckinghamshire, England) diluted 1:5000 in PBS-T for 1 hour at room temperature. The membranes were washed, incubated with chemiluminescent detection reagents (ECL-Plus, Amersham, Buckinghamshire, England) for 5 minutes at room temperature, and exposed to autoradiography film (X-OMAT, Kodak, Rochester, NY) to visualize the labeled proteins. Exposed films were scanned at 600 dots per inch, and relative levels of protein expression were determined by analyzing the pixel intensity of the bands using NIH Image software (Version 1.62) and normalized to actin or tubulin. For each data set, the experimental conditions were then normalized to control samples so that control was always set to 100%. The results from three independent sets of tissues (each set consisting of a control, presymptomatic, grade 1, and a grade 4 specimen) were pooled and assessed by one-way ANOVA for statistical significance followed by Fisher’s probable least-squares difference post-hoc test. For cells and Drosophila, protein levels were analyzed by Student’s t-test for statistical significance.
Real-Time Quantitative PCR
Following induction for 1, 3, and 5 days, PC12-74Q cells were harvested from 6 cm dishes and centrifuged at 12,000 g for 10 minutes. The pellets were resuspended in 1 ml of Trizol (Invitrogen, Carlsbad, CA), and RNA was extracted. The RNA was then purified according to the company’s protocol using the RNAeasy Mini kit (Qiagen Valencia, CA, USA). The total RNA concentration of each sample was then quantified by absorbance at 260 nm using an Ultraspec 3100pro (GE Healthcare, Piscataway, NJ). A total of 1 µg of RNA was converted to cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). PCR reactions were run in triplicate on an ABI Prism 7100 Sequence Detection System using 1.25 ng of RNA and 12.5 µl of Taqman Universal PCR MasterMix (Roche Diagnostics, Indianapolis, IN) per reaction. PCR primer pairs specific to rat profilin-1, rat profilin-2a, and rat ribosomal protein, S18 were obtained from Applied Biosystems. S18 was used as an endogenous control, and profilin transcript levels were calculated using the ΔΔCt method as per Applied Biosystems specifications. Four independent RNA extractions were tested.
Pulse-Chase Protein Labeling
Following two days of induction, PC12-74Q cells were incubated in DMEM lacking cysteine and methionine for 2 h at 37 °C with 10% CO2. The cells were then pulse-labeled in cysteine/methionine-free media containing 100 µCi/µl 35S-labeled cysteine/methionine ProMix (GE Healthcare, Piscataway, NJ, USA) for 30 minutes. After labeling, the cells were washed once with culture medium containing a 10-fold excess of unlabeled methionine and cysteine (2 mM each) and incubated further for the indicated times in media alone or media containing MG132 (10 µM) or rapamycin (2 µg/ml). Cells were collected at the different time points, and lysed in 200 µl RIPA buffer (50 mM Tris—HCl, 1% (v/v) Nonidet P-40, 0.1% (w/v) deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 1 × proteinase inhibitor, Complete Mini (Roche), and equal amounts of protein from the cleared lysates were immunoprecipitated with anti-profilin-1 and protein G-sepharose overnight at 4°C. The immune complexes were washed 3 times in RIPA buffer and separated on 16% SDS-PAGE. The gels were dried and exposed to a phosphorimager screen for visualization on a STORM phosphorimager system (Molecular Dynamics, Piscataway, NJ, USA).
The UAS-pros127Q flies used for these experiments have been previously described (Kazemi-Esfarjani and Benzer, 2002
). The UAS-chic line was the generous gift of Lynn Cooley (Yale University) and has also been previously described (Hopmann and Miller, 2003
). The GMR-gal4 and Elav-gal4 lines were supplied by K-T Min (NINDS). The flies were maintained on a standard mixture of corn meal, yeast, and agar at room temperature unless otherwise noted.
Newly eclosed flies were collected and reared on standard corn meal agar medium in a 25°C incubator. The flies were transferred to fresh media every 4 days, and the number of dead flies was counted daily. Differences in survival between the groups were examined using the log-rank test.
Phototaxis measurement was performed as described previously(Min and Benzer, 1999
). Shortly after eclosion, flies were placed in a 25°C incubator in the dark. On day 3, the flies were brought into a dark room and placed into a counter-current tube system. The flies were tapped gently to the bottom of the first tube and then the tube rack was placed horizontally with a light source at one end. The flies were allowed to move toward the light for 30 seconds, and those that did were then tapped down to the bottom of the next tube; this process was repeated six times. The number of flies in each tube was calculated as a percentage of the overall population tested. The mean ± standard error of five independent experiments was calculated for each fly strain; a total of 200 flies were tested per strain. Results between the groups were assessed by one-way ANOVA for statistical significance followed by Fisher’s probable least-squares difference post-hoc test for statistical difference between tubes for each fly strain.