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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00967

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Parkinson's disease -synuclein mutations exhibit defective axonal transport in cultured neurons

Anirban R. Saha^1,*, Josephine Hill^1,*, Michelle A. Utton¹, Ayodeji A. Asuni¹, Steven Ackerley¹, Andrew J. Grierson¹, Christopher C. Miller¹, Alun M. Davies², Vladimir L. Buchman², Brian H. Anderton¹ and Diane P. Hanger^1,

¹ Department of Neuroscience, PO Box 38, Institute of Psychiatry, King's College London, De Crespigny Park, London, SE5 8AF, UK
² Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, Edinburgh, EH9 1QH, UK

View larger version (40K): [in a new window]   Fig. 1. Distribution and transport of transfected -synuclein in neurons. (A) Rat cortical neurons 5 DIV were transfected with wild-type (WT) -synuclein and fixed at intervals after glycerol shock. Panels show the expression of exogenous -synuclein, detected with the 90 antibody, at 2.5 hours, 6 hours and 24 hours after glycerol shock. Cells fixed at 6 hours after glycerol shock are double labelled with 90 (left) and the antibody to tau, TP70 (right). Arrows indicate co-distribution of -synuclein and tau, arrowheads indicate dendritic extensions containing -synuclein. Scale bars, 50 µm. (B) Rat cortical neurons (5 DIV) were transfected with wild-type -synuclein and labelled with 90 at 30 minute intervals from 3 hours to 6 hours after glycerol shock. Neurons were either untreated (circles) or treated with the metabolic inhibitors sodium azide and 2-deoxy-D-glucose (triangles) between 3 hours and 3.5 hours after glycerol shock. Points represent mean distance travelled (in µm) from the axon hilum ± s.e.m.

View larger version (28K): [in a new window]   Fig. 2. -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 -synuclein and fixed 6 hours or 24 hours after transfection. -Synuclein expression was detected with 90. Arrows indicate the extent of -synuclein migration in the longest neurite extending from each neuron. Swellings containing A30P mutant -synuclein are indicated by arrowheads. Scale bars, 50 µm. (B) Human wild-type (circles), A30P (squares) and A53T (triangles) -synuclein were transfected into 5 DIV rat cortical cultures and labelled with 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 -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) -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) -synuclein. *P<0.001 compared with wild-type -synuclein. (D) Rat cortical neurons were transfected with human -synuclein plasmids, fixed 4 hours after glycerol shock and labelled with 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) -synuclein. The asterisk indicates P<0.05 for A30P -synuclein average immunofluorescence intensity compared with the wild type. Bars represent mean intensity of immunofluorescence ± s.e.m.

View larger version (39K): [in a new window]   Fig. 3. -Synuclein expression does not perturb axonal transport of mitochondria in rat cortical neurons. Rat primary neuronal cortical cultures (5 DIV) were transfected with a plasmid expressing the targeting sequence from subunit VIII of cytochrome c oxidase tagged with DsRed2 (MITO) or co-transfected with the mitochondrial marker plus EGFP-tagged wild-type (WT) or A30P mutant -synuclein (A30P), and fixed 24 hours after transfection. Protein expression was detected by direct fluorescence of DsRed2-cytochrome c oxidase (MITO), EGFP-tagged wild-type (WT) or A30P mutant (A30P) -synuclein. Scale bar, 50 µm.

View larger version (16K): [in a new window]   Fig. 4. Mutants that mimic permanent serine phosphorylation of -synuclein exhibit reduced transport in rat cortical neurons. -Synuclein mutants that mimic permanent states of phosphorylation (S87D, n=124 neurons, and S129D, n=56 neurons) or dephosphorylation (S87A, n=50 neurons, and S129A, n=53 neurons) of human -synuclein, or wild-type human -synuclein (WT, n=124 neurons) were transfected into 5 DIV rat cortical neurons. Bars represent mean rate of transport (µm hour–1) ± s.e.m. for each protein between 3 hours and 6 hours after glycerol shock. Transfection of the phosphorylation mimics of -synuclein, S87D or S129D, resulted in statistically significant reductions in the rate of transport compared to wild-type -synuclein (*P<0.001).

View larger version (34K): [in a new window]   Fig. 5. Replacement of tyrosine 125 in -synuclein by aspartate or phenylalanine does not affect -synuclein transport rate in rat cortical neurons. Rat cortical cultures transfected with plasmids expressing EGFP-tagged human wild-type or the Y125D or Y125F mutants of -synuclein, and their distributions were compared by direct fluorescence 4.5 hours after transfection. Scale bars, 50 µm. Human wild-type (black), Y125D (white) and Y125F (grey) -synuclein plasmids were transfected into 5 DIV rat cortical cultures. Bars indicate the mean rates of transport ± s.e.m. (µm hour–1) for each construct between 3 hours and 4.5 hours after glycerol shock (n=52-121 neurons).