Exercise increased α-synuclein concentration in blood plasma in 15-month old Y39C transgenic mouse.

<p>Mouse blood plasma samples were processed by Western blot analysis using antibodies to LB509 (<b>A</b>) and Syn-1 (<b>B</b>). Both monomer and dimer α-synucleins were found in mouse plasma. Sample images are shown for each Western blot. (<b>C, D</b>) Quantitative α-synuclein plasma levels are shown for each group after being normalized to mouse serum albumin. With both LB509 (<b>A</b>) and Syn-1 (<b>B</b>) antibodies, there were significant increases in α-synuclein monomer, dimer and total (monomer plus dimer) fractions in Exercise versus Non-Exercise mouse plasma (n = 7, t-test, *<i>p</i><0.05).</p

Exercise reduced α-synuclein oligomer formation in 15 month-old Y39C transgenic mouse brain.

<p>Brain tissues (cortex) from Exercise and Non-Exercise transgenic mice were analyzed for α-synuclein aggregation using LB509 (human only) and Syn-1 (mouse-plus-human) antibodies. (<b>A</b>) Western blots with LB509 show that exercise dramatically reduced α-synuclein oligomer formation in brain compared to mice of the same age not performing exercise. α-Synuclein monomer levels were not changed. The sample blots show Exercise (<b>Ex</b>) and Non-Exercise (<b>nEx</b>) animals. (<b>B</b>) Western blots with Syn-1 antibody show that Exercise mice had reduced α-synuclein oligomer and dimer fractions, as compared to Non-Exercise mice. (<b>C, D</b>) Western blot images were quantified. The relative levels of α-synuclein oligomer, monomer, and total (oligomer plus monomer) to β-actin are shown for Exercise and Non-Exercise transgenic mice. There were significant reductions in brain oligomer (n = 7, t-test, **<i>p</i><0.01) and total (n = 7, t-test, *<i>p</i><0.05) in the Exercise group compared to the Non-Exercise group.</p

Running wheel exercise in wild-type mice increased DJ-1 expression in muscle, plasma, and brain.

<p>Adult FVB/N wild-type mice (4–6 months old) were assigned to either an exercise group or a control group (n = 5 each). After one week of running wheel exercise, animals were sacrificed and tissues collected for DJ-1 protein analysis by Western blot (muscle and brain) and ELISA (plasma). (<b>A</b>) Quantitative data normalized to β-actin show that muscle DJ-1 levels were significantly higher in the exercise group than controls (t-test, **<i>p</i><0.01). (<b>B</b>) DJ-1 levels in plasma were significantly increased in exercise mice compared to control mice (t-test, **<i>p</i><0.01). (<b>C</b>) Brain DJ-1 levels were higher in exercise mice than control mice, but not significantly (t-test, <i>p</i> = 0.06). (<b>D</b>) Correlations between muscle and plasma DJ-1 (dashed line) and brain DJ-1 (solid line) in exercising mice. Both plasma and brain DJ-1 levels were significantly correlated with the exercise-induced change in muscle DJ-1 (plasma vs. muscle, <i>p</i> = 0.04 and brain vs. muscle, <i>p</i> = 0.03). All DJ-1 values are relative to non-exercise controls.</p

Exercise increased DJ-1, Hsp70 and BDNF levels in 15-month old Y39C transgenic mouse brain and also increased DJ-1 levels in muscle and plasma.

<p>(<b>A</b>) Brain tissues (cortex) were processed for Western blot analysis using antibodies to DJ-1, Hsp70, BDNF and β-actin after 3 months of running wheel Exercise (<b>Ex</b>) or no Exercise (<b>nEx</b>) in Y39C transgenic animals. Representative images are shown for all Western blots. (<b>B-D</b>) Quantitative protein levels in brain are shown for each group after being normalized to β-actin. There were significant increases in DJ-1, Hsp70 and BDNF proteins in Exercise mouse brain compared to Non-Exercise mice (n = 7, t-test, *<i>p</i><0.05, **<i>p</i><0.01). (<b>E)</b> Muscle samples were processed for Western blot analysis using DJ-1 and β-actin antibodies after 3 months of running wheel Exercise (<b>Ex</b>) or no Exercise (<b>nEx</b>) in control animals. (<b>F</b>) Muscle DJ-1 Westerns were quantified and are shown for each group after being normalized to β-actin. Muscle DJ-1 was significantly increased in Exercise mice compared to Non-Exercise mice (n = 7, t-test, **<i>p</i><0.01). (<b>G</b>) Plasma DJ-1 levels were measured by ELISA. Results show that the Exercise group had significantly higher plasma DJ-1 concentrations than the Non-Exercise mice (n = 7, t-test, **<i>p</i><0.01).</p

DJ-1 knockout mice had impaired performance on running wheels and on the Rotarod but normal cognitive and exploratory activity.

<p>Ten months old homozygous DJ-1 knockout (DJ-1 KO) mice and C57BL/6 wild-type (WT) littermates were trained to perform running wheel exercise in their individual cages. Daily running distances were recorded for two weeks. (<b>A</b>) Wild-type mice ran 5.57 ± 0.21 miles per day, while DJ-1 knockout mice were significantly slower, running 0.89 ± 0.06 miles per day (n = 6, multi-variance ANOVA test, **<i>p</i><0.01). Ten months old DJ-1 knockout mice and C57BL/6 wild-type littermates were also tested on the Rotarod (<b>B</b>), Morris water maze (<b>C</b>), and open field (<b>D</b>). (<b>B</b>) DJ-1 knockout mice fell from the Rotarod after a shorter period of time than WT mice when tested at 20 and 26 rpm (n = 6, multi-variance ANOVA, F_{(4, 60)} = 16.93, <i>p</i> = 0.0089, *<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) DJ-1 knockout mice and WT littermates had similar learning curves in the Morris water maze (n = 6, multi-variance ANOVA, <i>p</i>>0.1). (<b>D</b>) DJ-1 knockout mice and WT mice had similar exploratory activity in open field testing (n = 6, multi-variance ANOVA, <i>p</i>>0.1).</p

Y39C human mutant α-synuclein transgenic mice performed daily running wheel exercise for three months which led to improved motor and cognitive function.

<p>12-month-old Y39C transgenic mice were divided into Exercise and Non-Exercise groups (n = 7 for each group) following pre-testing of all 14 animals in individual cages with running wheels. Animals were assigned to Exercise and Non-Exercise groups by alternating rank order following their week-long pre-test. Exercise mice had free access to individual cage-mounted running wheels and Non-Exercise mice had a locked, non-functioning running wheel in individual cages. Daily running distances of the Exercise animals were recorded and averaged for each week. (<b>A</b>) Data show that all animals continued running for 12 weeks with some reduction in running speed. Average distance in the first week was 3.76 ± 0.87 miles per day. Average distance in the 12^th week was 2.71 ± 0.53 miles per day (no statistical difference between 1^st and 12^th week, n = 7, multi-variance ANOVA, <i>p</i> = 0.33). After 12-weeks of running wheel activity, all mice were tested for high intensity motor activity on the Rotarod (<b>B</b>) and cognitive function using a Morris water maze (<b>C</b>). (<b>B</b>) In the Rotarod test, the Exercise group could remain on the rod significantly longer at 26 rpm than the Non-Exercise group (n = 7, multi-variance ANOVA, **<i>p =</i> 0.001). (<b>C</b>) In the Morris water maze, the Exercise mice took significantly less time to find the hidden platform at Day 5 than Non-Exercise transgenic mice (n = 7, multi-variance ANOVA, *<i>p =</i> 0.02).</p

Growth properties of purified N27-A and unpurified N27 cells.

<p><b>(A-F):</b> Both cell types were plated at 20,000 cells in each well of 6-well plates. Representative images were taken at Day 1, 3, and 5 for each cell type. Purified N27-A cells (<b>Images A-C</b>) grew more slowly than unpurified N27 cells (<b>Images D-F</b>). <b>(G)</b>: Growth charts of purified N27-A (red line) and unpurified N27 cells (green line) from Day 0 to Day 7. Data present the average cell number from two wells of purified and unpurified cells in three experiments (n = 6, each cell type). Bar, 20 μm for <b>A-F</b>.</p

Process of N27 cells clonal purification.

<p><b>(A):</b> Phase contrast image of unpurified N27 cells grown at low density. <b>(B):</b> Immunostaining for TH with green fluorescence in unpurified N27 cells. Shown is a cluster of cells exhibiting bright TH-positive staining. Most cells shown in phase contrast have no TH-immunoreactivity. <b>(C):</b> Schematic drawing of the clonal culture procedures for purifying N27-A cells. Cells were plated at low density to form individual colonies which were picked up and screened in 48- and 96-well plates. The positive clones were expanded in 6-well plates and 10-cm dishes. Bar, 20 μm for both <b>A</b> and <b>B</b>.</p

Immunocytochemistry of purified N27-A and unpurified N27 cells for dopamine neuron markers TH and DAT.

<p>The N27 cells were cultured on 8-well chamber slides and immunostained for the dopamine neuron markers TH <b>(A-D)</b> and DAT <b>(E-H).</b> Other wells were double-stained for TH and the neuronal marker Tuj1 <b>(I-L).</b> To image every cell in each well, the nuclear marker DAPI was added to all wells. <b>(A-B):</b> The purified N27-A clone showed strong TH staining in all cells as demonstrated by dual-staining with TH and DAPI. <b>(C-D):</b> The unpurified N27 cell mixture revealed that only a small fraction of the DAPI-labeled cells were TH positive. <b>(E-F):</b> In the purified N27-A clone, all cells had moderate DAT staining as shown with DAT and DAPI double staining. <b>(G-H):</b> In the unpurified N27 cell mixture, very few cells were positive for DAT immunostaining. <b>(I-J)</b>: In the purified N27-A clone, all cells were double-positive for Tuj1 and TH. <b>(K-L)</b>: While there were few TH-positive cells in the unpurified N27 cell mixture, most cells were Tuj1 positive, demonstrating that the mixed cell population was neuronal. Bar, 50 μm for <b>A-L</b>.</p

Purified N27-A cells are more sensitive to 6-OHDA toxicity than unpurified N27 cells.

<p>Purified N27-A and unpurified N27 cells were cultured in 24-well plates (for trypan blue staining) and 96-well plates (for MTT assay). Two days after plating, cells were treated with 0–150 μM of 6-OHDA for 24 hr. The cell viability was measured by trypan blue staining <b>(A)</b> and MTT assay <b>(B)</b>. <b>(A):</b> Cell viability data from trypan blue staining showed that there were significantly fewer viable cells in purified N27-A cultures compared to unpurified N27 cultures. <b>(B)</b>: Cell viability results from the MTT assay also showed that purified N27-A cells had greater cell death than unpurified N27 cells after exposure to 6-OHDA. Reduced cell survival in both assays indicate that purified N27-A cells are more sensitive to 6-OHDA than unpurified N27 cells (n = 12 for <b>A</b>, n = 15 for <b>B</b>, **<i>p</i><0.01).</p