Human alpha -synuclein-harboring familial Parkinson's disease-linked Ala-53 right-arrow Thr mutation causes neurodegenerative disease with alpha -synuclein aggregation in transgenic mice -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Lee et al. (2002) Proc. Natl. Acad. Sci. USA 99 (13), 8968–8973. (10.1073/pnas.132197599)

Supporting Methods
Behavioral Assessment.
Behavioral monitor. An open-field monitor (TruScan, Coulbourn Instruments, Allentown, PA) was used to monitor total horizontal and vertical movements of mice. Following the habituation period, the mice were exposed to the monitor once a week for 2 h at the same time of the day. The movements of the mice were recorded as determined by interaction with two layers of photocells. Parameters measured include total number of moves, time spent moving, and also distance traveled in both the horizontal and vertical plane (rearing). The horizontal movements measure the general locomotor activity of the mouse and hence the general integrity of the motor circuits, whereas rearing is primarily related to the integrity of dopaminergic nervous system. Accelerating rotarod test
. Rotorod test was performed using Rotamax 4 (Columbus Instruments, Columbus, OH). The mice were trained to stay on the rotating drum using a mild electric shock when they fall off. Following the training period, the mice were tested twice a week using a paradigm where the rotational speed was increased from 5 to 25 rpm over a 7-min period. The time at which the mice fall off the rod is recorded. This is repeated three times a day in the evening at least 2 h prior to ‘night’ at half-hour intervals, once a week. The mean latency time of the three trials is recorded. Initially the mice are ‘trained’ on the rod by two consecutive days of three trials. This test studies the integrity of cerebellar function as well as the lower motor unit function. HPLC analysis of striatal dopamine and dopamine metabolites
. Striatum from one half of the brain was analyzed for monoamine neurotransmitters and metabolites by high performance liquid chromatography with electrochemical detection at +0.240 mV essentially as described (1). Dopamine, and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA) and serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were separated and detected in a single chromatogram. Samples were sonicated in 250 m l of 0.1 M HClO₄. Duplicate aliquots (20 m l) of the resulting homogenates were reserved for protein analysis and the remaining amounts centrifuged at 7,200 ´ g (12,000 rpm) for 10 min. 50 m l of each supernatant was injected onto a 100 by 4.6 mm 3 m m C₁₈ reversed-phase chromatography column in a mobile phase containing 0.1 M monochloroacetic acid, 5-9% acetonitrile, 0.4–0.5 g/liter octanesulfonic acid, 0.3–0.4% triethylamine and 100 m g/liter EDTA. 5-Hydroxy-N-methyltryptamine was used as the internal standard. Relative peak areas of the sample peaks were compared to external standards for quantitation. Protein was measured by the method of Lowry et al. (2).

1. Adrews, A. M., Ladenheim, B., Epstein, C. J., Cadet, J. L. & Murphy, D. L. (1996) Mol. Pharmacol. 50, 1511–1519.

2. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265–275.