Abstract Exercise has been recommended to improve motor function in Parkinson patients, but its value in altering progression of disease is unknown. In this study, we examined the neuroprotective effects of running wheel exercise in mice. In adult wild-type mice, one week of running wheel activity led to significantly increased DJ-1 protein concentrations in muscle and plasma. In DJ-1 knockout mice, running wheel performance was much slower and Rotarod performance was reduced, suggesting that DJ-1 protein is required for normal motor activity. To see if exercise can prevent abnormal protein deposition and behavioral decline in transgenic animals expressing a mutant human form of α-synuclein in all neurons, we set up running wheels in the cages of pre-symptomatic animals at 12 months old. Activity was monitored for a 3-month period. After 3 months, motor and cognitive performance on the Rotarod and Morris Water Maze were significantly better in running animals compared to control transgenic animals with locked running wheels. Biochemical analysis revealed that running mice had significantly higher DJ-1, Hsp70 and BDNF concentrations and had significantly less α-synuclein aggregation in brain compared to control mice. By contrast, plasma concentrations of α-synuclein were significantly higher in exercising mice compared to control mice. Our results suggest that exercise may slow the progression of Parkinson’s disease by preventing abnormal protein aggregation in brain.

Citation: Zhou W, Barkow JC, Freed CR (2017) Running wheel exercise reduces α-synuclein aggregation and improves motor and cognitive function in a transgenic mouse model of Parkinson's disease. PLoS ONE 12(12): e0190160. https://doi.org/10.1371/journal.pone.0190160 Editor: Richard Jay Smeyne, Thomas Jefferson University, UNITED STATES Received: August 2, 2017; Accepted: December 8, 2017; Published: December 22, 2017 Copyright: © 2017 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper. Funding: The study was supported by The Walter S. and Lucienne Driskill Foundation, the Leopold Korn and Michael Korn Professorship in Parkinson’s Disease, and by the personal savings of C.R.F. Competing interests: The authors declare no conflict of interest. Abbreviations: BDNF, brain derived neurotrophic factor; Ex, exercise; Hsp70, heat shock protein 70; LB509, antibody specific for human α-synuclein; nEX, non-exercise; RPM, revolutions per minute; Syn-1, antibody recognizing both mouse and human α-synuclein; Tg, transgenic; Y39C, tyrosine replaced by cysteine at residue 39 of human α-synuclein

Introduction Parkinson’s disease is characterized by the loss of midbrain dopamine neurons in the substantia nigra pars compacta [1, 2]. Death of dopamine neurons has been attributed to oxidative stress, abnormal protein aggregation, and genetic factors [3–5]. Mutations in many genes have been linked to Parkinson’s including α-synuclein, Parkin, UCHL1, DJ-1, PINK1, LRRK2, and VSP35 [6–8]. The gene mutations could lead to either loss of neuroprotective functions such as DJ-1 and PINK1, or gain of toxic functions such as α-synuclein and LRRK2 [9]. Single amino acid mutations in the α-synuclein gene as well as simple triplication of the gene have been shown to cause autosomal-dominant forms of Parkinson’s disease [10, 11]. Interestingly, α-synuclein has been found to be a major component of Lewy bodies which are pathological hallmarks for idiopathic Parkinson’s disease [12, 13]. Transgenic mice overexpressing human wild-type or A53T mutant α-synuclein have recapitulated many features of Parkinson’s disease, such as impaired motor function, abnormal protein aggregation, and neuronal degeneration [14–17]. Monomeric α-synuclein protein is highly soluble; however, it can easily aggregate under various conditions such as low pH, high concentration, presence of metal ions, and oxidative stress. The formation of insoluble α-synuclein fibrils involves several intermediate species, such as dimers, oligomers, and protofibrils. Recent evidence suggests that α-synuclein oligomers are the most neurotoxic form of α-synuclein protein, and oligomer secretion is critical for the spreading and progression of Parkinson’s neuropathology [18–20]. Therefore, preventing α-synuclein aggregation could provide a major therapeutic advance [21, 22]. DJ-1 is one of the Parkinson-associated genes in which mutations lead to early-onset, autosomal recessive disease. Because the loss of gene expression causes disease, the DJ-1 gene can be seen as protecting nearly everyone from developing Parkinson’s disease [23–25]. DJ-1 or its homologs are present in all life forms that use oxygen including all animals, all plants that perform photosynthesis, and all aerobic bacteria [26–29]. This critical gene protects cells by antioxidant mechanisms such as stabilizing Nrf2 (nuclear factor erythroid 2-related factor) and thereby upregulating a family of antioxidant response element (ARE) genes [30–32]. DJ-1 is also involved in regulating HIF1 transcriptional activity under hypoxic conditions [33]. We have shown that DJ-1 also protects cells from abnormal protein aggregation by upregulating Hsp70 [34, 35]. Because Parkinson’s disease leads to disabling bradykinesia and rigidity, exercise and physical therapy are often prescribed by physicians. The hope has been that exercise will enhance mobility, preserve muscle tone, and prevent medical complications such as pneumonia that are associated with immobility. Several clinical trials have found that regular exercise or physical therapy may improve motor function in Parkinson patients [36–39]. In acute, drug-induced animal models of Parkinson’s disease, exercise can partially protect dopamine neurons from neurotoxicity [40–45]. For Alzheimer’s disease, exercise in transgenic mouse models have shown improvement in cognitive function and reduction in β-amyloid deposition as well as other biochemical markers [46–50]. However, in transgenic mouse models of Huntington’s disease, results of exercise testing are conflicting; either accelerating disease progression [51] or partially improving motor and cognitive function [52–54]. In this report, we have found that running wheel exercise can be neuroprotective in transgenic mice which have a progressive, age-related form of a Parkinson’s-Plus, diffuse Lewy body disease.

Materials & methods Animals All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Denver. Mice with DJ-1 gene deletion (B6.129-Park7tm1Mak, abbreviated as DJ-1 KO mice) were kindly provided by Dr. Tak Mak (University of Toronto) [55]. The homozygous DJ-1 KO mice and wild-type C57BL/6J littermates were produced by breeding male and female heterozygous DJ-1 KO mice. The Y39C human α-synuclein transgenic mice (FVB-Tg(Thy1-SNCA*Y39C)5Crf, abbreviated as Y39C a-Syn Tg mice) have been described previously [56]. The animals express a human mutant form of α-synuclein (Y39C) in all neurons under control of the Thy-1 promoter and so are a model of diffuse Lewy body disease, a Parkinson-Plus disorder. The Y39C a-Syn Tg mice were bred through male heterozygous Y39C Tg mice with wild-type FVB/N female mice. Heterozygous Y39C Tg mice and wild-type FVB/N littermates were used for this study. For all running wheel studies, mice were individually housed in each cage with free access to food and water. For all experiments, 5 to 7 animals were used for each group. We tried to use both males and females equally if possible for each group. Running wheel exercise in mice Animals were singly housed in cages with free access to a running wheel (12 cm in diameter) mounted on the food bin. Daily running distances were recorded with a bicycle odometer attached to the running wheel. Before testing the Y39C-human mutant α-synuclein transgenic strain, we first tested 6-month old FVB/N wild-type mice for one week to evaluate the distance traveled by normal mice having access to a running wheel. There were 5 wild-type mice each for the running group and for the control group, with 3 males and 2 females in each group. To see the effect of the DJ-1 gene on locomotor activity, we then tested mice with the DJ-1 gene knocked out (6 DJ-1 KO mice, 3 males and 3 females) and compared the distances they ran with wild-type C57BL/6 littermates (6 wild-type controls, 3 males and 3 females). Both groups were 10-months of age. These animals had access to running wheels for two weeks. For our Y39C human mutant α-synuclein transgenic mice, we tested 12-month old mice for their ability to exercise on running wheels for one week. A total of fourteen Y39C Tg mice were rank-ordered by their running distance during that one week and were assigned to long term exercise group (seven Y39C Tg mice, 4 males and 3 females) or to non-exercise group (seven Y39C Tg mice, 4 males and 3 females) in alternating rank order. The exercise group had free access to running wheels in each of their cages for three months, while the non-exercise group had locked, non-functioning running wheels in their cages. Weekly running wheel distances were recorded using a bicycle odometer for each exercising animal. Rotarod test Mice were tested for their ability to run on a 3 cm diameter rotating rod (Rotarod) at speeds ranging from 3 to 33 rpm [35, 56]. Before the test, mice were trained to stay on the Rotarod at 3 rpm. During 5 testing days, mice were placed on the rotating rod at one of the pre-set speeds of 7, 14, 21, 28, or 33 rpm for a 5-minute trial. Each animal received 3 trials with 5-minute rest intervals between trials. The time the mice spent on the Rotarod without falling was recorded for each trial. Morris water maze testing Spatial learning was assessed using the Morris Water Maze in our campus animal behavioral core [35, 56]. The maze included a circular tank (120 cm in diameter) filled to 10 cm below the edge of the tank with 27°C water that was made opaque by the addition of non-toxic black ink. A circular escape platform (10 cm in diameter) was located 1 cm below the surface of the water in a constant location in the northwest quadrant of the tank. Mice were first acclimated to the maze during three trial habituation sessions. Each testing session consisted of 4 consecutive days with four trials per day. The platform was invisible in the pool, and mice were allowed to swim for 60 seconds before being returned to the home cage. The time to find the platform from all training and testing sessions was collected. Open field testing Mice were placed in a 1-meter square plastic box for 30 minutes of open field exploratory testing. The field was divided into central and peripheral areas. The exploratory paths were recorded for each animal via video camera and computer. The time that mice spent in central and peripheral areas was analyzed. The rearing events of animals were also recorded. Western blotting The mouse brain and muscle tissues were dissected and homogenized in dissociation buffer with protease inhibitors [35, 56]. Blood plasma was prepared as described below. Protein concentrations were determined by the BCA method. Fifty μg of protein was separated on 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The blots were probed with antibodies to DJ-1 (1:5000, #AB9718, Millipore), α-synuclein (Syn-1, 1:2000, #610787; BDBioscience) and α-synuclein (LB509, 1:2000, #MABN824, Millipore), Hsp70 (1:2000, #AB9920, Millipore), BDNF (1:2000, #SAB2108004, Sigma), β-actin (1:4000, #A2228, Sigma), and mouse serum albumin (1:2000, #PA1-30899, Thermo Fisher Scientific). Blots were incubated with HRP-conjugated secondary antibodies (1:10,000; #115-035-003, #111-035-003, Jackson Immuno Research), followed by chemiluminescent detection. Protein densities were quantified by ImageJ software after scanning into image files and normalized to β-actin or mouse albumin. ELISA Mouse blood was collected immediately after sacrifice through transcardiac needles and syringes containing EDTA as an anti-coagulant. The blood was centrifuged at 2000 rpm for 5 min. After centrifugation, the plasma fraction was collected and stored at -80°C. Plasma was used to determine DJ-1 concentrations using an ELISA kit (CircuLex), according to manufacturer’s instructions. Statistics For behavioral tests and biochemical analyses, there were 5–7 animals per group. The number of animals for each group was determined by power analysis using our previous behavioral and biochemical data. Data were expressed as mean ± SEM. Data were analyzed using t-test or ANOVA test followed by the Fisher LSD post hoc test.

Discussion We have discovered that a functional DJ-1 gene is required for normal, voluntary running wheel performance in mice. In young wild-type mice as well as in aging transgenic mice expressing mutant human α-synuclein in all neurons, running wheel exercise can increase DJ-1 protein levels in muscle, plasma, and brain. We have found that long term running wheel exercise has a neuroprotective effect in our transgenic mice. Exercise significantly improves motor and cognitive function while dramatically reducing α-synuclein oligomer accumulation in brain while increasing plasma concentrations of α-synuclein. The mechanism by which exercise leads to these beneficial effects appears to be related to upregulation of DJ-1 and other neuroprotective factors such as Hsp70 and BDNF in the brain. We and others have reported that increased expression of DJ-1 can render neurons more resistant to oxidative stress and to misfolded protein accumulation [34, 58, 59]. Using in vitro experiments in N27 dopamine neurons, we have demonstrated that increased DJ-1 protein levels can protect neurons from oxidative stress by increasing glutathione production through upregulation of the rate limiting step in glutathione synthesis. If, instead, the cell stress is overexpression of mutant human α-synuclein, increased levels of DJ-1 do not change glutathione synthesis but do increase expression of Hsp70 [34]. In the current study, the exercise-induced increase in DJ-1 and Hsp70 in brain are likely preventing the formation of α-synuclein oligomers. Other researchers have shown that exercise can increase neurogenesis in hippocampus, increase BDNF expression, and improve memory function in various mouse models [60–65]. Treadmill running can activate the anti-oxidant master gene Nrf2 and protect mice from the neurotoxic effects of MPTP [40, 44, 66]. In the Alzheimer transgenic mouse model, exercise can decrease β-amyloid concentrations by the activation of SIRT-1 signaling pathway [50]. There has been no previous investigation of long-term exercise in transgenic models of Parkinson’s disease. Our results indicate that long-term exercise can prevent the development of age-related neurodegeneration in a transgenic mouse model of diffuse Lewy body disease, a Parkinson-Plus disorder. In humans, diffuse Lewy body disease is a currently untreatable form of Parkinson’s. Because exercise produces sweeping changes in all aspects of physiology from sensorimotor activity to lipid metabolism in muscle, it is difficult to define a hierarchy of beneficial effects on brain function. Since mice which lack the DJ-1 gene cannot perform on running wheels or on the Rotarod with the same intensity as wild-type animals, DJ-1 appears to be essential for dealing with the physiological stress created in muscle by sustained motor activity. Because DJ-1 knockout animals have the same cognitive performance as wild-type mice in the Morris Water Maze and on open field exploration, the DJ-1 deficit does not appear to influence cognition nor low intensity motor activity. To precisely define the role of muscle verse brain derived DJ-1, organ-specific DJ-1 knockouts would have to be developed. Our study gives insight into the mechanism by which exercise prevents α-synuclein oligomer accumulation in brain. While oligomer formation was reduced in brains of mice with access to running wheels, the same animals showed increased plasma concentrations of α-synuclein monomers and dimers. α-Synuclein is known to be present in plasma of humans and other mammals, but the exact source of plasma α-synuclein remains uncertain. While it is possible that red blood cells may release α-synuclein into plasma, the protein may come from central and peripheral neurons [67, 68]. Our findings in Y39C transgenic mice show that plasma α-synuclein comes from neurons rather than red blood cells because plasma α-synuclein is approximately 50:50 human/mouse mixture as is brain. By contrast, red blood cell α-synuclein is 100% mouse in our Y39C animals. α-Synuclein has been found in plasma exosomes as a soluble protein [69, 70]. Studies have shown that α-synuclein can be secreted from cultured neurons via exosomes [71, 72]. We have recently shown that lysosomal and exosomal genes are activated in vitro by overexpression of mutant human α-synuclein as well as by DJ-1. Activated lysosomes and exosomes are the likely route of enhanced α-synuclein secretion from the cytosol to the plasma (Cummiskey, Zhou, Freed et al., unpublished data). In the same transgenic mouse strain used in the current study, we have shown that the drug phenylbutyrate can increase DJ-1 levels, prevent α-synuclein oligomer formation in brain, and stop age-related decline in motor and cognitive function [35]. Exercise appears to have the same neuroprotective effects as phenylbutyrate. Our earlier in vitro experiments showed that the beneficial effects of phenylbutyrate were dependent on the expression of the DJ-1 gene. Blocking DJ-1 expression with anti-sense RNA blocked the drug’s ability to protect neurons from oxidative stress and from misfolded protein. This result indicates that DJ-1 is required to exert the neuroprotective effect of phenylbutyrate [35]. Because we have found that DJ-1 knockout mice have much reduced spontaneous activity on running wheels and have impaired performance on the Rotarod, the DJ-1 protein appears to be essential for normal motor function. Since DJ-1 knockout animals have normal swimming and cognitive abilities in the Morris Water maze as well as normal exploratory behavior in open field testing, it is likely that their reduced activity on the running wheel or the Rotarod represents a limitation in maximal muscle performance rather than in cognitive function. In summary, we have found that voluntary exercise on a running wheel can upregulate DJ-1 in muscle and brain of a transgenic mouse model of Parkinson’s disease and can prevent the age-related decline of motor and cognitive abilities normally seen in this transgenic strain. Since we have described similar beneficial effects with the drug phenylbutyrate in these transgenic mice, we hypothesize that patients with Parkinson’s disease might be able to slow or stop disease progression from either an intensive exercise program or treatment with the drug phenylbutyrate.

Acknowledgments The animal behavioral tests were done at Animal Behavioral Core, the Center for Neuroscience, University of Colorado Denver.