Mulberry fruit ameliorates Parkinson's-disease-related pathology by reducing α- synuclein and ubiquitin levels in a 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine/probenecid model  Pil Sung Gu, Minho Moon, Jin Gyu Choi, Myung Sook Oh  Journal of Nutritional Biochemistry  Volume 39, Pages 15-21 (January 2017) DOI: 10.1016/j.jnutbio.2016.08.014 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 Experimental design for treatment of ME, MPTP/p, and vehicle and behavioral analysis. p.o., per os; T: test. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Protective effect of ME against MPTP/p-induced olfactory dysfunction in mice. Saline or 250 mg/kg ME was administered orally once per day for 38 days, and mice received a total of 10 injections of MPTP (25 mg/kg, i.p.) in combination with probenecid (100 mg/kg, i.p.) 5 days after first administration of ME. The vertical axis of the graph represents the latency time to find the buried food pellet in the cage. Values are given as the mean±S.E.M. #P<.05, ##P<.01 and ###P<.001 compared with the normal group; ⁎⁎P<.01 compared with the MPTP/p group. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 Protective effect of ME against MPTP/p-induced motor impairments in mice. Saline or 250 mg/kg ME was administered orally once per day for 38 days, and mice received a total of 10 injections of MPTP (25 mg/kg, i.p.) in combination with probenecid (100 mg/kg, i.p.) after 5 days of ME administration. The latency to fall off the rotarod was recorded over three trials per mouse in rotarod test (a). The time that mice needed to turn down completely was recorded as the T-LA in pole test (b). The total distance traveled was measured over 30 min in open field test (c). Values are given as the mean±S.E.M. #P<.05, ##P<.01 and ###P<.001 compared with the normal group; ⁎P<.05, ⁎⁎P<.01 and ⁎⁎⁎P<.001 compared with the MPTP/p group. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 Protective effect of ME against MPTP/p-induced loss of dopaminergic neurons. Dopamine neurons were visualized with TH immunostaining. The optical density in the ST (a) and the number of TH-positive neurons in the SNpc (b) were measured. Representative photomicrographs were taken of the ST (c–e) and the SNpc (f–h). (c and f) Normal group; (d and g) MPTP/p group; (e and h) MPTP/p+ME group. Each column represents the mean±S.E.M. Data are expressed as percentages relative to untreated normal. #P<.05 compared with the normal group; ⁎P<.05 and ⁎⁎P<.01 compared with the MPTP/p group. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 Inhibitory effect of ME on MPTP/p-induced overexpression of α-synuclein and ubiquitin. Saline or 250 mg/kg ME was administered orally once per day for 38 days, and mice received a total of 10 injections of MPTP (25 mg/kg, i.p.) in combination with probenecid (100 mg/kg, i.p.) after 5 days of ME administration. The α-synuclein level was assessed by Western blot analysis using α-synuclein antibodies in the whole protein of the SN (a) and ST (b). The ubiquitin level was assessed by Western blot analysis using ubiquitin antibodies in the whole protein of the SN (c) and ST (d). Values are given as the mean±S.E.M. #P<.05 and ##P<.01 compared with the normal group; ⁎P<.05 compared with the MPTP/p group. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 6 Proposed therapeutic mechanisms of mulberry fruit in PD. Mulberry fruit directly inhibits MPTP/p-induced up-regulation of α-synuclein and ubiquitin, the markers for LBs. The death of dopaminergic neurons is attenuated by mulberry fruit via antioxidant and antiapoptotic actions in the animal models of PD. Based on the present study, it can be speculated that the neuroprotective effects of mulberry fruits may be mediated by inhibition of LBs formation. In addition, the mulberry fruit significantly inhibits both motor and nonmotor dysfunction in models of PD. Journal of Nutritional Biochemistry 2017 39, 15-21DOI: (10.1016/j.jnutbio.2016.08.014) Copyright © 2016 Elsevier Inc. Terms and Conditions