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Fig. 2. ${alpha}$ -Synuclein mutants associated with Parkinson's disease exhibit reduced transport in neurons. (A) Rat primary neuronal cortical cultures were transfected at 5 DIV with wild-type (WT), A53T or A30P ${alpha}$ -synuclein and fixed 6 hours or 24 hours after transfection. ${alpha}$ -Synuclein expression was detected with ${alpha}$ 90. Arrows indicate the extent of ${alpha}$ -synuclein migration in the longest neurite extending from each neuron. Swellings containing A30P mutant ${alpha}$ -synuclein are indicated by arrowheads. Scale bars, 50 µm. (B) Human wild-type (circles), A30P (squares) and A53T (triangles) ${alpha}$ -synuclein were transfected into 5 DIV rat cortical cultures and labelled with ${alpha}$ 90 at 3 hours, 4 hours, 5 hours and 6 hours after glycerol shock. Points represent mean distance travelled (in µm) from the axon hilum ± s.e.m. The differences between the distances moved by wild-type and mutant ${alpha}$ -synucleins were statistically significant at each time point examined [P<0.05 at 3 hours, P<0.01 at 4 hours, 5 hours and 6 hours after glycerol shock (n=40-124 neurons)]. (C) ${alpha}$ -Synuclein was transfected into 5 DIV rat cortical cultures as above and rates of transport were calculated at hourly intervals during the time of observation. Bars show mean rates ± s.e.m. for wild-type (black), A30P (white) and A53T (grey) ${alpha}$ -synuclein. ^*P<0.001 compared with wild-type ${alpha}$ -synuclein. (D) Rat cortical neurons were transfected with human ${alpha}$ -synuclein plasmids, fixed 4 hours after glycerol shock and labelled with ${alpha}$ 90. The average intensity of immunofluorescence within the neuronal cell body was measured for wild-type (n=96 neurons), A30P (n=138 neurons), and A53T (n=82 neurons) ${alpha}$ -synuclein. The asterisk indicates P<0.05 for A30P ${alpha}$ -synuclein average immunofluorescence intensity compared with the wild type. Bars represent mean intensity of immunofluorescence ± s.e.m.

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