Lactulose and Melibiose Attenuate MPTP-Induced Parkinson’s Disease in Mice by Inhibition of Oxidative Stress, Reduction of Neuroinflammation and Up-Regulation of Autophagy

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DAergic) neurons in the ventral brain. A disaccharide trehalose has demonstrated the potential to mitigate the DAergic loss in disease models for PD. However, trehalose is rapidly hydrolyzed into glucose by trehalase in the intestine, limiting its potential for clinical practice. Here, we investigated the neuroprotective potential of two trehalase-indigestible analogs, lactulose and melibiose, in sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with MPTP generated significant motor deficits, inhibited dopamine levels, and down-regulated dopamine transporter (DAT) in the striatum. Expression levels of genes involved in anti-oxidative stress pathways, including superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (NRF2), and NAD(P)H dehydrogenase (NQO1) were also down-regulated. Meanwhile, expression of the oxidative stress marker 4-hydroxynonenal (4-HNE) was up-regulated along with increased microglia and astrocyte reactivity in the ventral midbrain following MPTP treatment. MPTP also reduced the activity of autophagy, evaluated by the autophagosomal marker microtubule-associated protein 1 light chain 3 (LC3)-II. Lactulose and melibiose significantly rescued motor deficits, increased dopamine in the striatum, reduced microglia and astrocyte reactivity as well as decreased levels of 4-HNE. Furthermore, lactulose and melibiose up-regulated SOD2, NRF2, and NQO1 levels, as well as enhanced the LC3-II/LC3-I ratio in the ventral midbrain with MPTP treatment. Our findings indicate the potential of lactulose and melibiose to protect DAergic neurons in PD.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DAergic) neurons in the ventral brain. A disaccharide trehalose has demonstrated the potential to mitigate the DAergic loss in disease models for PD. However, trehalose is rapidly hydrolyzed into glucose by trehalase in the intestine, limiting its potential for clinical practice. Here, we investigated the neuroprotective potential of two trehalase-indigestible analogs, lactulose and melibiose, in sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with MPTP generated significant motor deficits, inhibited dopamine levels, and down-regulated dopamine transporter (DAT) in the striatum. Expression levels of genes involved in anti-oxidative stress pathways, including superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (NRF2), and NAD(P)H dehydrogenase (NQO1) were also down-regulated. Meanwhile, expression of the oxidative stress marker 4-hydroxynonenal (4-HNE) was up-regulated along with increased microglia and astrocyte reactivity in the ventral midbrain following MPTP treatment. MPTP also reduced the activity of autophagy, evaluated by the autophagosomal marker microtubuleassociated protein 1 light chain 3 (LC3)-II. Lactulose and melibiose significantly rescued motor deficits, increased dopamine in the striatum, reduced microglia and astrocyte reactivity as well as decreased levels of 4-HNE. Furthermore, lactulose and melibiose up-regulated SOD2, NRF2, and NQO1 levels, as well as enhanced the LC3-II/LC3-I ratio in the ventral midbrain with MPTP treatment. Our findings indicate the potential of lactulose and melibiose to protect DAergic neurons in PD.

INTRODUCTION
Parkinson's disease (PD), characterized by resting tremor, rigidity, bradykinesia, and postural instability, is a common neurodegenerative disease in the elderly (Jankovic, 2008). The pathological studies find a massive loss of dopaminergic (DAergic) neurons in the pars compacta of the substantia nigra (Surmeier et al., 2017). The neurodegeneration of PD could be caused by a complex interaction of genetic and environmental factors (Kalia and Lang, 2015). Genetic mutations involved in the oxidative stress pathway, such as synuclein alpha (SNCA), parkin RBR E3 ubiquitin-protein ligase (PRKN), Parkinsonism associated deglycase (DJ1), PTEN induced kinase 1 (PINK1) and leucine-rich repeat kinase 2 (LRRK2), are reported in patients with familial PD (Dias et al., 2013;Zuo and Motherwell, 2013). Genetic variants in glucosylceramidase β (GBA), proved to be the main risk for developing PD (Murphy et al., 2014), affecting autophagy activities (Aharon-Peretz et al., 2004;Gan-Or et al., 2015). A variety of environmental insults, including pesticides and 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), specifically increase oxidative stress, damage DAergic neurons and produce parkinsonism similar to the main features to PD (Tuite and Krawczewski, 2007), although only prolonged chronic but not acute or sub-acute MPTP exposure in mice triggers the formation of α-synuclein inclusion pathology (reviewed in Konnova and Swanberg, 2018). Therefore, compounds that reduce oxidative stress and up-regulate autophagy may be therapeutic strategies for PD patients.
Trehalose, a disaccharide found in plants and animals, demonstrates the potential to assist protein folding during environmental stress (Elbein et al., 2003). In cell and rodent models of Alzheimer's disease (AD), trehalose protects neurons by reducing aggregation of Aβ and could be a therapeutic candidate for AD (Liu et al., 2005;Du et al., 2013). Trehalose also demonstrates neuroprotective potential in other aggregationprone neurodegenerative diseases such as Huntington's disease (Tanaka et al., 2004), amyotrophic lateral sclerosis (Castillo et al., 2013) and spinocerebellar ataxia (SCA) type 17 (Chen et al., 2015). Neuroprotective and anti-neuroinflammatory effects of trehalose were also observed in a chronic MPTP-induced PD mouse model (Sarkar et al., 2014). Also, trehalose could accelerate the clearance of mutant huntingtin/α-synuclein (Sarkar et al., 2007), TATA-box binding protein  and ataxin 3 (Lin et al., 2016) by enhancement of autophagy. However, trehalose is rapidly hydrolyzed by trehalase in the intestine (Dahlqvist, 1968), limiting its application for disease treatment.
Previously two trehalase-indigestible analogs, lactulose, and melibiose were found to up-regulate autophagy in aggregationassociated SCA type 3 and 17 cell models Lin et al., 2016). In the present study, we examined the neuroprotective potential of trehalose and these two disaccharides in the MPTP-induced PD mouse model. Our findings provide new drug candidates for PD via up-regulating anti-oxidative stress and autophagy pathways as well as reducing neuroinflammation.

Sub-chronic MPTP Mouse Model
The animal experiments were conducted following the guidelines and were approved by the National Taiwan Normal University (NTNU) Research Committee. Male C57BL/6 mice (8 weeks old, 18-22 g) were purchased from the National Laboratory Animal Center (Tainan City, Taiwan). The mice were kept in individually ventilated cages under controlled temperature (25 ± 2 • C), relative humidity (50%), and 12 h on/off light cycle with ad libitum access to food and water at the Animal House Facility of NTNU.
After 2-week habituation, mice were randomly divided into five groups (n = 8). Regular drinking water or drinking water with 2% trehalose, lactulose, or melibiose was applied to the mice for 42 days. Experimental parkinsonism was established by i.p. injections of 15 total doses of MPTP (30 mg/kg in 0.9% saline; Toronto Research Chemicals, Toronto, ON, Canada) along with probenecid (250 mg/kg in 0.1 M NaOH; Sigma-Aldrich), while the control group received injections of saline. Probenecid was administered 1 h before MPTP administration as it decreases the clearance of MPTP and intensifies its neurotoxicity (Lau et al., 1990). The 15 dose regimen was administered over 3 weeks with five doses per week (once daily for five consecutive days, see flow chart in Figure 1A). Appropriate guidelines were abided in handling MPTP. The water was changed once a week and mouse body weight, blood glucose, and drinking amount were monitored every week for 4 weeks. There was no notable difference in terms of mouse body weight, blood glucose, and drinking amount among these five groups. Behavioral analyses were performed during the period to evaluate the treatment effect.

Behavioral Test
The pole test is a practical method to detect the degree of bradykinesia in the PD mouse model (Ogawa et al., 1985). Mice were placed head down on top of a vertical wooden pole (diameter 8 mm, height 50 cm), which was wrapped in gauze to prevent slipping (Yang et al., 2011). The time it took for the mice to climb down with all four feet on the floor was measured. Each mouse was required to perform three successive trials at 5 min interval. This test was performed at days 14, 21, 28, 35, and 42 (see flow chart). All the mice were pre-trained three times before the formal tests.
Also, stride length was measured in a gait test (Klapdor et al., 1997). To obtain footprints, the front and back paws were painted with nontoxic red and blue paints, respectively. Mice were allowed to walk along a narrow, paper-covered corridor (50 × 10 cm) toward a goal box, and stride length were measured manually as the distance between two paw prints using a digital vernier caliper. This test was performed on day 42, and the average of three strides was taken for each animal.

HPLC Analysis of Dopamine
Levels of dopamine in the striatum were determined by high-performance liquid chromatography (HPLC) analysis. Briefly, the isolated brain striatum was homogenized in 500 µl of PRO-PREP TM protein extraction solution (iNtRON Biotechnology Inc., Gyeonggi-do, Korea). The samples were centrifuged at 10,000× g for 30 min and then filtered through a 0.45 µm syringe membrane. Dopamine from the supernatant was analyzed by the HPLC system using a C18 column with a UV detector at 254 nm. The sample was passed through the HPLC system using a mobile phase of 87.5% 90 mM of sodium phosphate, 40 mM of citric acid, 10 mM of octane sulfonic acid, 3 mM of ethylenediaminetetraacetic acid and 12.5% acetonitrile (pH 3.0) at a flow rate of 1.0 ml/min.

Statistical Analysis
For each set of values, three independent experiments were performed and data were expressed as the means ± standard deviation (SD). Differences between groups were evaluated by student's t-test or ANOVA followed by an LSD post hoc test where appropriate. All P-values were two-tailed, with values of P < 0.05 considered significant.

Effects of Trehalose, Lactulose, and Melibiose on MPTP-Induced Motor Behavior in Mice
MPTP, a prodrug to the neurotoxin MPP + which selectively destroys DAergic neurons in the brain, was frequently applied to establish a mouse model for PD (Blandini and Armentero, 2012). MPTP treatment in mice also down-regulated autophagy and increased the level of α-synuclein, while enhancement of autophagy reduced the loss of DAergic neurons (Liu et al., 2013). Given that trehalose could up-regulate autophagy and demonstrate neuroprotective potential in MPTP-treated mice (Sarkar et al., 2007(Sarkar et al., , 2014, we established a sub-chronic MPTP mouse model (Figure 1A) to examine the neuroprotective effects of trehalose and its analogs lactulose and melibiose ( Figure 1B) on PD. Trehalose is formed by a 1,1-glycosidic bond between two α-glucose units. Lactulose is a synthetic disaccharide comprising fructose and galactose. It is produced by the isomerization of lactose with chemical or enzymatic methods (Aider and de Halleux, 2007). Melibiose exists in natural plants such as cacao beans and is formed by an α-1, (C) Immunohistochemistry of TH (red) and 4-HNE (green) positive neurons in ventral midbrain with MPTP/trehalose/lactulose/melibiose treatment. Nuclei were counter stained with 4 ,6-diamidino-2-phenylindole (DAPI; blue). Percentage of dopaminergic neurons with oxidative damages, based on TH and 4-HNE co-localization, were shown below (n = 8). P-values, ANOVA with LSD post hoc test, MPTP vs. control ( # P < 0.05, ## P < 0.01 and ### P < 0.001) and disaccharide-treated vs. untreated (*P < 0.05, **P < 0.01 and ***P < 0.001).
6 linkage between galactose and glucose. In the pole test, before MPTP administration (day 14), there were no differences in the time of landing between the five groups (control group: 6.0 ± 0.6 s, MPTP group: 5.9 ± 0.9 s, trehalose-treated group: 5.8 ± 0.6 s, lactulose-treated group: 6.0 ± 0.3 s, and melibiose group: 5.9 ± 0.6 s; P > 0.05), indicating the presence of similar baselines for all groups (Figure 2A). After neurotoxin injection, MPTP-treated mice showed a marked motor deficit (24-27% increase of landing time) as compared with the control group (5.4 ± 0.4 s vs. 4.3 ± 0.5 s at day 35, 5.7 ± 0.7 s vs. 4.5 ± 0.7 s at day 42; P < 0.001). On the other hand, mice with trehalose treatment displayed recovery (4.3 ± 0.7 s at day 35, P < 0.01; 4.1 ± 0.6 s at day 42, P < 0.001) in comparison to mice with MPTP only. Moreover, treatment of lactulose or melibiose also exhibited significant improvement on landing time (decrease of time to Nuclei were counter stained with DAPI (blue). Percentages of GFAP + cells and fluorescent intensity of GFAP were shown below (n = 8). P-values, ANOVA with LSD post hoc test, MPTP vs. control ( # P < 0.05 and ## P < 0.01) and disaccharide-treated vs. untreated ( * P < 0.05 and * * P < 0.01).

DISCUSSION
Increased oxidative stress and decreased antioxidant capacity including reduced SOD and increased 4-HNE are among pathological findings in postmortem brains of human PD and the MPTP-induced PD mouse model (Yoritaka et al., 1996;Castellani et al., 2002;Sofic et al., 2006;Li and Pu, 2011;Lv et al., 2012). Recently, in vitro studies showed that treatment with trehalose significantly reduced oxidative stress induced by chloroquine or cadmium via activating the NRF2 pathway, suggesting its strong anti-oxidant effect (Mizunoe et al., 2018;Wang et al., 2018). It is important to note that trehalose is readily digested by trehalase in the gut of humans (Dahlqvist, 1968), which implicates trehalaseindigestible analogs rather than trehalose as the potential treatments for aggregation-associated neurodegenerative disease. Here, we demonstrated the anti-oxidative and neuroprotective effects of two trehalase-indigestible analogs, lactulose, and melibiose, in the MPTP-induced PD mouse model. Although the elevations of striatal dopamine levels by lactulose and melibiose may be lower compared with trehalose, both of them still demonstrate improvements of motor deficits similar to trehalose. Furthermore, lactulose and melibiose increased DAT, SOD2, NRF2, and NQO1, and decreased 4-HNE, IBA1, and GFAP in the ventral midbrain of MPTP-induced PD mice. These findings suggest that lactulose and melibiose, similar to trehalose, may exert their anti-oxidative and anti-neuroinflammatory capacity to provide neuroprotection. Consistent with our findings, Sarkar et al. (2014) also demonstrate that trehalose can reduce the activation of microglia and astrocytes in the MPTP-induced PD mouse model.
Lines of evidence implicate targeting autophagy as a potential PD therapeutic strategy (Moors et al., 2017;Zhu et al., 2019). The depletion of autophagy gives rise to neurotoxicity accumulation and causes the loss of nerve cells (Hara et al., 2006;Komatsu et al., 2006). It has been proved that α-synuclein fibrils or aggregates are cleared by the autophagy-lysosomal pathways (Bae et al., 2014). Moreover, PD-associated proteins including LRRK2 (Orenstein et al., 2013;Manzoni et al., 2016), PINK1 (Lazarou et al., 2015), PRKN (Narendra et al., 2008) and ATP13A2 (ATPase cation transporting 13A2; Bento et al., 2016) are involved in autophagy-processing modulation as well. As an autophagy inducer, trehalose has the therapeutic potential on cellular and animal models of aggregation-prone neurodegenerative diseases (Sarkar et al., 2007;Rodríguez-Navarro et al., 2010;Casarejos et al., 2011;Lan et al., 2012;Schaeffer et al., 2012;Lee et al., 2015;Lin et al., 2016). In SCA17 and SCA3 cell models, we found that lactulose and melibiose demonstrate anti-aggregation and neuroprotection effects mainly through autophagy-activation Lin et al., 2016). Our results showed MPTP treatment down-regulated autophagy function by reducing the conversion of LC3-II from LC3-I. Similar to trehalose, lactulose and melibiose increased the ratio of LC3-II/LC3-I in the ventral midbrain of MPTP-treated mice, suggesting their potential to up-regulate autophagy in PD.
This study demonstrates the neuroprotective potential of lactulose and melibiose in the MPTP-induced PD mouse model, by activating NRF2 and autophagy pathways. However, their neuroprotective effects may not be better than trehalose, even though they are trehalase-indigestible. Although not broken down by human enzymes, lactulose and melibiose can be metabolized in the colon by Bifidobacterium, Lactobacillus or Saccharomyces species (Ostergaard et al., 2000;Bouhnik et al., 2004;De Souza Oliveira et al., 2011), which may lead to less concentration of lactulose and melibiose in the brain. Further investigations to refine their metabolism by intestinal flora of microorganisms will be necessary to enhance their neuroprotective effects.
In conclusion, our results show that lactulose and melibiose reduce motor deficits, inhibit the loss of striatal dopamine, increase DAT, decrease 4-HNE level, reduce activation of microglia and astrocytes, and enhance anti-oxidative and autophagy functions in the ventral brain of MPTP-induced PD mice. Future studies in different PD models will be warranted to confirm their potentials as treatments for human PD.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article.

ETHICS STATEMENT
The animal study was reviewed and approved by National Taiwan Normal University (NTNU) Research Committee.