Charvin et al. 10.1073/pnas.0502698102. |
Fig. 6. (A) Lack of tyrosine hydroxylase (TH) expression in striatal cell cultures. Total proteins were extracted as described in ref. 1 from mouse primary striatal cells (E14) cultured for 7 days (cultures) and total striatum (tissue). Western blots were processed in parallel for TH (1/1,000) (Insitut Jacques Boy, Reims, France), glutamate decarboxylase (Rabbit pan GAD, 1/1,000) (Chemicon) and tubulin (a-tubulin, clone DM1A, 1/10,000) (Sigma). Note the lack of TH expression in cultures when compared to the tissue. Note also the equal quantity of GAD in both extracts. (B) Kinetics of c-Jun activation in transfected striatal cells. P-c-Jun-positive neurons were detected in transfected neurons as in Fig. 2 and 3. Note the slight but significant increase in expanded huntingtin (expHtt) but not Htt-expressing neurons as soon as 8 h after transfection. Note also that this increase occurs when aggregates are not yet in the nucleus.
1. McLaughlin, B. A., Nelson, D., Erecinska, M. & Chesselet, M. F. (1998) J. Neurochem. 70, 2406-2415.
Supporting Methods
Primary Striatal Cell Cultures.
Embryos were removed at day 14 from timed-pregnant Swiss mice (Janvier, Le Genest-St-Isle, France) or D2 receptor knockout mice. Striata were dissected and mechanically dissociated by gently pipetting in neurobasal medium (GIBCO/BRL) as previously described (1). After decantation for 5 min at room temperature to eliminate tissue debris, cells were collected by centrifugation at 1,000 ´ g for 5 min. Cell pellets were resuspended in neurobasal medium containing 1´ B27 supplement (Invitrogen), 500 mM L-glutamine (Invitrogen), 100 units/ml penicillin-streptomycin (Invitrogen) and 25 mM 2-mercaptoethanol (Sigma). Cells were then plated at a density of 1,000 cells per mm2 into Nunc multiple-well plates coated with 50 mg/ml poly-D-lysine (Sigma). Cells were cultured in the complete neurobasal medium at 37°C with 5% CO2. After 3 days, cell culture medium was changed, and all experiments were performed 7 days after dissection, when most of the cells were of neuronal phenotype and postmitotic.Transient Expressions and Treatments.
cDNAs for huntingtin (Htt) and expanded Htt (expHtt) were provided by the Huntington’s Disease Foundation Resource Bank (University of California, Los Angeles). pcDNA3.1 expression plasmids containing the entire exon 1 sequence for the human huntingtin gene (IT15), with either 25 or 103 continuous CAA or CAG repeats, were engineered behind a CMV promoter. A sequence encoding an EGFP was inserted in frame at the C terminus of each construct. In parallel, the coding sequence of the EGFP gene was expressed under the control of the CMV promoter (referred to as GFP). Lipofectamine 2000 reagent was used to transfect cDNA plasmids by following the protocol provided by the manufacturer (Invitrogen). After 4 h of transfection, the medium was removed and replaced by the complete neurobasal medium containing dopamine (DA; Calbiochem) or quinpirole (Research Biochemicals, Natick, MA), then cells were replaced at 37°C for the appropriate time. For the pharmacological treatments, 10 mM SCH-23390 (Sigma), 1 mM raclopride (Sigma), 200 mM ascorbate (Aldrich), or 20 mM SP-600125 (Calbiochem) were added 30 min before and during DA treatment.Immunofluorescence Experiments.
Cultures were fixed in 2% paraformaldehyde in the culture medium for 40 min. Fixed cells were then permeabilized with methanol/acetone (1:1) for 10 min at 4°C, rinsed three times in PBS, and blocked in 10% normal goat serum in PBS for 1 h at room temperature. Cells were then incubated with primary antibodies in PBS containing 1% BSA overnight at 4°C: monoclonal anti-microtubule-associated protein 2 [anti-MAP2, kindly provided by B. Riederer (Institut d’Anatomie, Lausanne, Switzerland)] (1:100), polyclonal anti-phospho Ser-73 c-Jun (1:500, Ozyme), and polyclonal anti-phospho Thr-183/Tyr-185 JNK (1:100, Ozyme). After washing with PBS, cells were incubated with (Cy3)-conjugated anti-rabbit IgG (1:2,000, Amersham Pharmacia) or (Cy3)-conjugated anti-mouse IgG (1:2,000, The Jackson Laboratory) for 2 h at room temperature. Double labeling experiment of P-c-Jun and MAP2 was performed by using anti-P-c-Jun and anti-MAP2 antibodies and was revealed with a (Cy3)-conjugated anti-rabbit IgG and a biotinylated horse anti-mouse IgG (1:100, Vector Laboratories), respectively. MAP2 immunoreactivity was visualized by using 7-amino-4-methylcoumarin-3-acetic acid (AMCA) Avidin D (1:100, Vector Laboratories) for 30 min at room temperature. Samples were finally counterstained with Hoechst, mounted with VECTASHIELD mounting medium (Vector Laboratories), and observed under a Leica DM LB fluorescence microscope.TUNEL Assay and Death Measurement.
DNA fragmentation was detected by the TUNEL staining according to the manufacturer’s instructions (Roche Molecular Biochemicals) with minor modifications. Briefly, cells were fixed, permeabilized, and counterstained with Hoechst as describe above. The cells were then covered with 50 ml of TUNEL mixture for 1 h at 37°C in a humidified chamber. After three washes in PBS, samples were mounted with VECTASHIELD mounting medium (Vector Laboratories).Neurons were also scored as surviving or dying cells by morphological criteria based on the DNA labeling with Hoechst. Neurons containing condensed or fragmented nuclei were scored as dying cells. Data are expressed as the percentage of surviving cells in total counted cells.
Statistical Analysis.
Results are presented as means ± SEM. For each condition, at least three independent experiments were performed. Each experiment was realized in duplicate (two independent wells), and a minimum of 100 transfected cells were counted per well, meaning 600 transfected neurons per experimental condition.For P-c-Jun induction, P-c-Jun immunoreactivity was quantified with the image analysis software IMAGE PRO PLUS 4.5.0.19 (Media Cybernetics). A basal threshold was established from the intensity of P-c-Jun immunoreactivity in Htt-transfected neurons and then applied on P-c-Jun immunoreactivity in expHtt-transfected neurons. Neurons with a P-c-Jun immunoreactivity more intense than this threshold were counted as P-c-Jun positive cells.
Results were compared by using ANOVA between subjects, and post hoc comparisons were made using the indicated test (see Figs. 3–5). All analyses were done with WINDOWS STATVIEW 5.0 software (Abacus Concepts, Berkeley, CA).
1. Garcia, M., Charvin, D. & Caboche, J. (2004) Neuroscience 127, 859–870.