Dopaminergic Loss and Inclusion Body Formation in α-Synuclein Mice: Implications for Neurodegenerative Disorders
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Takeda A., et al., Am. J. Pathol. 152, 367 (1998);
Wakabayashi K., Matsumoto K., Takayama K., Yoshimoto M., Takahashi H., Neurosci. Lett. 239, 45 (1997).
A 1480–base pair (bp) fragment of 5′-flanking region of the human PDGF-β chain gene was isolated from the psisCAT plasmid (a gift from T. Collins Harvard Medical School) and placed upstream of a Not I–Sal I fragment consisting from 5′ to 3′ of an SV40 splice 423 bp of human cDNA encoding full-length wild-type α-synuclein and SV40 sequence from the pCEP4 vector (Invitrogen) providing a polyadenylate signal. The resulting fusion gene was freed of vector sequences purified and microinjected into one-cell embryos (C57BL/6 × DBA/2 F 2 ) according to standard procedures. Thirteen transgenic founders were identified by slot-blot analysis of tail DNA and bred with wild-type C57BL/6 × DBA/2 F 1 mice to establish transgenic lines. Transgenic offspring were identified by polymerase chain reaction (PCR) analysis of tail DNA. Genomic DNA was extracted and amplified in 30 cycles (93°C for 30 s 57°C for 30 s 72°C for 1.5 min) with a final extension at 72°C for 5 min. Primers were as follows: 5′-CCAGCGGCCGCTCTAGAACTAGTG (sense) and 5′-CCAGTCGACCGGTCATGGCTGCGCC (antisense).
E. Masliah et al. data not shown.
Double-immunolabeling studies were performed on 40-μm-thick vibratome sections which were first incubated overnight at 4°C with a human α-synuclein–specific antibody (1:1000) (Fig. 1 D to F) followed by detection with the Tyramide Signal Amplification-Direct (Tyramide Red) system (1:100; NEN Life Sciences Boston MA). Sections were then incubated overnight with a monoclonal antibody (mAb) to TH (1:10; Roche Molecular Biochemicals) followed by incubation with a fluorescein isothiocyanate (FITC)– conjugated secondary antibody to mouse immunoglobulin G (IgG) (1:75; Vector Laboratories). The specificity of the primary antibodies was confirmed in control experiments in which sections were incubated with preimmune serum instead of primary antibody or with primary antibody preabsorbed for 48 hours with a 20-fold excess of the peptide to which the antibody was raised or in the absence of primary antibody. Other sections were double-immunolabeled with antibody to human α-synuclein (as above) and rabbit polyclonal antibody to ubiquitin (1:50 or 0.2 mg/ml; DAKO Corporation Carpinteria CA) detected with an FITC-conjugated secondary antibody to rabbit IgG (1:75; Vector Laboratories). To evaluate the integrity of presynaptic terminals and dopaminergic neurons we double-immunolabeled sections with a mAb to synaptophysin (1:2500; Roche) (Tyramide Red detection system) and a mAb to TH (see above). Brain sections from mice to be compared in any given experiment were processed and immunolabeled in parallel. Three sections were analyzed per mouse and four serial 2-μm-thick optical sections were obtained per section. For each experiment the linear range of the intensity of immunoreactive structures in control sections was determined with a MRC1024 (Bio-Rad) confocal microscope. This setting was then used for the collection of all images to be analyzed in the same experiment. Digitized images were transferred to a Power-PC Macintosh computer and NIH Image 1.4 software was used to calculate the percent image area covered by immunoreactive terminals. The number of TH-positive neurons in the pars compacta of the substantia nigra was estimated essentially as described [
Mice were evaluated as described (19) with a rotorod (San Diego Instruments San Diego CA). Initially they were trained for five trials. During the subsequent test trials mice were placed individually on the cylinder and the speed of rotation increased from 0 to 40 rpm over a period of 240 s. The length of time mice remained on the rod (fall latency) was recorded and used as a measure of motor function.
Masliah E., Neurosci. News 1, 14 (1998).
For Western blot analysis of TH levels brains were homogenized and separated into cytosolic and particulate fractions as described (4). Twelve micrograms of cytosolic fraction per mouse were loaded onto 10% SDS–polyacrylamide gel electrophoresis gels followed by transfer of proteins onto Immobilon membranes and detection of TH with a mAb to TH (1:1000; Roche). Blots were incubated with horseradish peroxidase–coupled secondary antibody and developed with the chemiluminescence reagent (NEN). After exposure of blots to film the density of bands was quantitated with the ImageQuant system (Molecular Dynamics Sunnyvale CA).
We thank S. Ordway and G. Howard for editorial assistance and M. Alford for technical assistance. Supported by NIH Grants AG5131 and AG10689 (to E.M.) and AG11385 (to L.M.) and the Spencer Family Foundation (to M.H.).