Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder accompanied by movement deficits with selective degeneration of dopaminergic neurons in the substantia nigra (SN). Recent studies indicate that early diagnosis of PD has important implications for the disease-modifying strategy for PD showing not only some dopaminergic neuronal damage but also non-motor symptoms, which occur several years before the onset of motor symptoms. However, studies on the relationship between non-motor symptoms and its underlying mechanisms from the early to the late phase of PD are unknown. Here, we aimed to show alterations in the nonmotor symptoms of PD, including colonic dysmotility and impaired olfaction, and the related factors by intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) plus probenecid (MPTP/p). A mouse model of the early stage of PD was developed by systemic administration of MPTP (25 mg/kg,i.p.) and probenecid (100 mg/kg,i.p.) at 3.5-day intervals for a total of 10 injections. We performed motor and non-motor behavioral tests after 3 (called asymptomatic) and 10 (called symptomatic) injections of MPTP/pcompared with the untreated (called control) group. We found that there were motor disturbances at the symptomatic stage, while impairments in intestinal motility and olfaction were observed from the asymptomatic stage. We also found the reduction of dopaminergic neuronal cell numbers in the SN and striatal dopamine transporter levels starting from the asymptomatic stage. At both asymptomatic and symptomatic stages, we demonstrated alterations in the expression of several proteins that are associated with non-motor deficits in the mouse ileum or olfactory bulb compared with the control group. Our findings in chronic MPTP/p-induced mice suggest their potential use as an animal model for the early stage of PD as well as a significant correlation between changes in relevant factors and symptoms.

1. Introduction

Parkinson’s disease (PD), the second most common neurodegenerative disease, is characterized by motor deficits such as resting tremor, muscle rigidity, bradykinesia, and gait disturbance [1, 2]. These PD motor symptoms are caused by selective dopaminergic neuronal damage in the substantia nigra (SN) of the brain [3, 4]. Although the diagnosis of PD is entirely dependent on clinical motor symptoms caused by a dopamine deficiency, various prodromal non-motor symptoms such as several dysfunctions of the gastrointestinal (GI),olfactory, autonomic, and psychiatric systems arise at least 10 years before the onset of motor impairment [5-7]. It has been reported that autonomic dysregulations like drooling and increased heart rates as well as neuropsychological symptoms like sleep disturbances, mood disorders, and cognitive impairment are common features in early PD [8-10]. Among these non-motor deficits, impairments of the GI and olfactory functions are the most prevalent and predictable premotor symptoms experienced in approximately 60-70% of PD patients prior to the onset of motor signs [11]. Given this fact, precise diagnosis of PD non-motor symptoms is crucial for the development of disease-modifying therapies for PD. Therefore, there is great need for an animal model of PD that presents early non-motor symptoms as well as clinical motor symptoms.

Accumulated studies on preclinical models that can reproduce the symptoms and pathology of early PD have been reported by either genetic mutation or treatment with chemical neurotoxins such as 6hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). For example, mice with mutations in the leucine-rich repeat kinase 2 gene, which is one of the mutation forms in familial PD, exhibited olfactory loss and anxiety symptoms, but did not have dopaminergic neuronal damage in the SN [12]. Mice overexpressing human wild type α-synuclein under the Thy1 promoter displayed colonic motor dysfunction with no significant alterations in the related proteins, including choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase, and vasoactive intestinal peptide, in the colon [13].In addition, it has been reported that alterations in the mRNA levels of phosphatidylcholine lipids or brain-derived neurotrophic factors in the SN were regarded as a pre-symptomatic marker following intracerebral injection of 6-OHDA into the SN of mice [14, 15]. Mice receiving
intraperitoneal injections of MPTP also showed potential as an experimental model of the early stage of PD because of changes in the transcriptome profile of SNCA and the dopamine receptor 2 gene or activity of monoamine oxidase B in the SN and striatum (ST) [16, 17]. In particular, chronic administration of a low dose of MPTP with probenecid (MPTP/p) induced motor deficits accompanied by dopaminergic neuronal damage, gliosis, and α-synuclein inclusion in the SN [18]. However, these studies could not reproduce the non-motor symptoms and mainly focused on alterations in the brain due to genetic or neurotoxic insults.In this study, we hypothesized that the most frequent non-motor events of PD, such as dysfunctions of the GI and olfactory systems, could be reproduced by MPTP/p administration. Moreover, we explored the underlying mechanisms, including changes in the regulatory proteins that are related to intestinal motility and olfaction from the early to the late phase of PD induced by MPTP/p.

2. Materials and methods
2.1. MPTP/probenecid-induced PD model

Male 10-week-old C57BL/6 mice were purchased from Central Lab Animal Inc. (Seoul, South Korea). The animals were housed (n = 4 per cage, 2 cages per group) at an ambient temperature of 23 ± 1 °C and relative humidity of 60 ± 10% under a 12-h light/dark cycle and were allowed free access to water and food. This model was established according to our previously reported methods [19]. Mice were divided into 3 groups according to the number of MPTP/p administrations: Group 1, control (without MPTP/p injection); Group 2, asymptomatic (3 injection times of MPTP/p); Group 3, symptomatic (10 injection times of MPTP/p). Briefly, all mice except the control group were injected with MPTP hydrochloride (25 mg/kg/day in saline, i.p.) along with probenecid (100 mg/kg/day in 5% NaHCO3, i.p.). Probenecid was administered 30 min prior to MPTP injection as it induces a chronic PD model via reducing the clearance of P505-15 inhibitor MPTP and increasing the rate of passage to the blood-brain barrier [20-22]. Mice received a total of 10 injections of MPTP in combination with probenecid at an interval of 3.5 days.

2.2. Behavior test
2.2.1. Open field test

The open field test is a useful method to measure ambulation ability in mice [23]. We performed the test between 9 p.m. and 2 a.m. to avoid diurnal variation. Mice were placed in the testing chamber (40 × 25 × 18 cm) with white floors, followed by a 30-min recording period using a computerized automatic analysis system (Biobserve, Germany). The data collected by computer included the total distance traveled by tracking the center of the animal.

2.2.2. Rotarod test

The rotarod test is a useful method for measuring motor coordination in a mouse model of PD [24]. The rotarod unit consists of a rotating spindle (7.3 cm diameter) and five individual compartments. After two times of training (8-10 rpm rotation speed), the rotation speed was increased to 12 rpm in a test session. The time each mouse remained on the rotating bar was recorded over two trials per mouse with a maximum length of 3 min per trial. Data are presented as the mean time on the rotating bar over the two trials.

2.2.3. Bead expulsion test

The bead expulsion test was performed for monitoring intestinal motility [25, 26]. A plastic bead (diameter, 3 mm) was inserted into the colon at a distance of 2 cm from the anal verge. The time required for expulsion of the bead was measured and taken as an estimate of intestinal motility.

2.2.4. Buried pellet test

The buried pellet test is a useful method to examine olfactory deficits in the early stages of PD [27, 28]. Mice were food-deprived for 20 h before the test. The test was conducted in a clean plastic cage (24 × 42 × 15 cm). A cheese-smelly pellet was buried 1 cm under the bedding in a cage corner, and the mouse was positioned in the center of the cage. The time spent to bite the pellet was measured at a maximum trial length of 5 min per mouse.

2.3. Tissue preparation

The mice were sacrificed after all behavior tests were finished at 3 and 10 injections of MPTP/p, respectively. For immunohistochemical studies, the mice were anesthetized and transcardially perfused with 0.05 M PBS, and then fixed with cold 4% paraformaldehyde (PFA) in a 0.1 M phosphate buffer. The brains were quickly removed and postfixedin a 0.1 M phosphate buffer containing 4% PFA overnight 4 °C and then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Serial coronal sections that were 30 μm-thick were cut on a freezing microtome (Leica, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at 4 °C. For western blotting analysis, the mice were decapitated and the ileum tissues were isolated and stored at −80 °C until use.

2.4. Western blotting

Ileum tissues were lysed with a triple-detergent lysis buffer. The lysates were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and gels were processed for antigens and blotted onto immobilon-P transfer membranes for 1 h 30 min. Membranes were incubated with 5% skim milk in a mixture of tris-buffered saline and Tween 20 for 1 h and then with the primary antibodies (TH 1:1500, inducible nitric oxide synthase (iNOS) 1:1500, α-synuclein 1:2000, and β-actin 1:3000) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. Blots were detected using an enzyme-linked chemiluminescence detection kit, and an LAS-4000 mini system (Fujifilm Corp., Japan) was used for visualization. The intensities of the bands were normalized to the β-actin band using Multi Gauge software (Fujifilm Corp., Japan).

2.5. Immunohistochemistry

Brain sections were taken from the each brain region: between bregma − 3.16 mm and bregma − 3.64 mm (SN), between bregma 0.98 mm and bregma 0.38 mm (ST), and between bregma 4.28 mm and bregma 3.56 mm (olfactory bulb, OB) according to the mouse brain atlas [29]. The brain sections were briefly rinsed in PBS and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a rabbit anti-TH antibody (1:1000) for SN and OB, a rabbit antidopamine transporter (DAT) antibody (1:500) for ST, and a goat antiChAT (1:100) for OB tissues overnight at 4 °C in the presence of 0.3% triton X-100. After rinsing in PBS, the sections were then incubated with biotinylated anti-rabbit and anti-goat IgG (1:200) for 1 h,and with an avidin-biotin complex mixture (1:100) for 1 h at room temperature. Peroxidase activity was visualized by incubating sections with 3,3diaminobenzidine in a 0.05 M tris–buffer. After several rinses with PBS, the sections were mounted on gelatin-coated slices, dehydrated, and cover-slipped using a slide mounting medium. To examine microgliosis in the OB, the brain sections were washed with PBS and incubated with goat anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody (1:1000) overnight at 4 °C in the presence of 0.3% triton X-100. After rinsing in PBS, the sections were incubated with chicken anti-goat Alexa 488 (1:500) for 1 h and then 4′,6-diamidino-2-phenylindole staining was performed for 20 min. The immunofluorescent sections were mounted with an anti-fade fluorescent medium (Wako chemical, Japan). The images were acquired at 200× or 400× magnifications using an optical light microscope (BX51; Olympus, Japan) equipped with a 20× objective lens.

2.6. Measurements of optical density and the number of immunoreactive cells

For measurement of the optical density of TH-, DAT-, or ChAT-positive areas in the ST or OB, the total region of interest was manually outlined and averaged optical densities were acquired in images with converted eight-bit indexed color. The number of THor Iba1-positive cells was calculated according to stereological counting [30]. The images were analyzed with Image J software.

2.7. Statistical analysis

All statistical analyses were conducted using the software GraphPad Prism Version 5.0. Values are expressed as the mean ± standard error of mean (SEM). All data were evaluated by Student’s t-test. Differences with a p-value < 0.05 were considered statistically significant.

3. Results and discussion

In the present study, we demonstrated that mice treated with MPTP in combination with probenecid exhibited the dysfunctions of intestinal motility and olfaction as non-motor symptoms of PD and its underlying mechanisms from the asymptomatic to the symptomatic stages of PD.First, we performed two motor behavior tests to investigate alterations in movement functions by MPTP/p injections. We found that locomotor activity in the open field test was significantly impaired in the symptomatic group (6546.04 ± Autoimmune vasculopathy 76.40 cm) compared with the control group (10,746.26 ± 523.50 cm; Fig. 1A). In the rotarod test, motor ability was significantly reduced in the symptomatic group (38.20 ± 15.25 s) compared with the control group (121.67 ± 7.98 s; Fig. 1B). These results show that motor symptoms are revealed at the symptomatic stage, but not at the asymptomatic stage.Several studies reported that the reduction in striatal DAT levels occurs concurrently with dopaminergic neuronal loss prior to the onset of motor symptoms inpatients with early PD [31–34]. We explored how brain dopaminergic neuronal density and DAT levels are altered by MPTP/p injections. The number of dopaminergic neuronal cell bodies in the substantia nigra pars compacta (SNpc) were remarkably reduced starting from the asymptomatic stage (27841.27 ± 2040.66 cells/ mm3) to the symptomatic stage (14821.43 ± 1820.94 cells/mm3) compared with the control group (33,333.33 ± 1343.86 cells/mm3;Fig. 2A). The percentage of striatal DAT levels compared to the normal group was also significantly decreased starting from the asymptomatic stage (54.15 ± 9.84%) to the symptomatic stage (50.23 ± 6.64%) in accord with the results in Fig. 2A (Fig. 2B). These changes due to MPTP/p administration in nigrostriatal brain regions indicate a pathological status similar to that of the asymptomatic stage of PD patients.

We explored changes in the non-motor features such as GI dysmotility and hyposmia after MPTP/p injections. In the bead expulsion test to examine intestinal dysmotility, the time to expel the bead was significantly longer at the asymptomatic stage (443.59 ± 31.33 s) than that of the control group (326.00 ± 40.42 s; Fig. 3A). Impaired olfaction as indicated by the cheese-pellet retrieval time was also observed starting after the asymptomatic group (83.73 ± 15.25 s) compared with control group (32.45 ± 8.45 s; Fig. 3B). These data indicate that two non-motor symptoms of PD are reproduced starting at the early stage in a PD mouse model induced by MPTP/p.Intestinal motility is regulated by enteric dopaminergic neurons as well as by nitric oxide in enteric macrophages [35–37]. In addition, αsynuclein overexpressing transgenic mice exhibited abnormal intestinal motility, indicating that overexpression of α-synuclein is involved in GI disturbance [38, 39]. To examine whether several factors that regulate intestinal motility are altered at the asymptomatic stage of PD induced by MPTP/p, we analyzed the expression levels of TH, iNOS, and αsynuclein in the ileum, an important intestinal region for modulating intestinal motility, between the asymptomatic and the control mice [40]. The ratio of TH expression levels in the ileum of the asymptomatic mice (0.66 ± 0.09) was significantly reduced while that of the symptomatic mice (0.95 ± 0.12) was nearly the same compared with the control mice (Fig. 4A). Each ratio of ileal iNOS and α-synuclein expression levels was significantly enhanced on the asymptomatic (iNOS; 2.40 ± 0.36, α-syn; 1.39 ± 0.10) and symptomatic (iNOS; 1.67 ± 0.17, α-syn; 1.61 ± 0.14) groups compared with the control group (iNOS; 0.95 ± 0.13, α-syn; 1.00 ± 0.04), respectively (Fig. 4B, C).Next, we explored whether the
immunoreactivity of ChAT and the number of Iba1-positive cells are altered in the OB of mice with severe olfactory deficits at the asymptomatic stage of PD.Witt and colleagues reported that
impaired olfaction in the early phase of PD may be closely associated with damage to the OB rather than alterations in the olfactory epithelium because biopsy tissues from the olfactory epithelium of PD patients are normal [41]. Another study showed that intranasal administration of MPTP can cause olfactory dysfunction with the injury in olfactory epithelium, not in OB [42]. These previous reports indicate the importance of using MPTP/p-induced mice to reflect olfactory impairment in the early stage of PD patients. In addition, it has been reported that cholinergic neuronal loss and excessive microgliosis in the OB region were observed in PD patients with impaired olfaction or MPTP-treated monkeys [43, 44]. Thus, severe cholinergic neuronal damage and microglial activation in the OB could cause the olfactory symptoms at the early stage of PD. The percentage of cholinergic neuronal density in the external plexiform layer of the OB was significantly reduced in the asymptomatic (74.72 ± 6.32%) and symptomatic (65.67 ± 7.51%) groups while that in the glomerular layer (GL) of the OB (asymptomatic group, 102.65 ± 5.27%; symptomatic group, 89.42 ± 8.94%) showed no difference compared with the control group (Fig. 4D). These results coincide with the outcomes of early PD patients [43]. We also found a significant increase in Iba1positive microglial cells of the asymptomatic (12014.78 ± 505.62 cells/mm3) and symptomatic (11820.65 ± 818.37 cells/mm3) groups in the GL of the OB compared with the control group (9420.64 ± 652.14 cells/mm3; Fig. 4E). In support of these findings, a previous report by Seo and colleagues found that excessive microgliosis was observed in the OB of a murine model of Niemann-Pick disease associated with PD [44]. Otherwise, loss of dopaminergic neurons in the OB of asymptomatic and symptomatic mice was not observed (Supplementary Fig. 1). This finding is consistent with the results of a previous report by Ferrer et al. who found that dopaminergic cells were rarely changed in the OB of PD patients [45]. A recent neurochemical study also exhibited that the levels of several monoamine neurotransmitters like dopamine, serotonin, and noradrenaline were little changed in the OB of MPTP-treated monkeys at all stages [46]. Meanwhile, it has been reported that an increased number of TH-positive dopaminergic cells were present in the OB of PD patients according to a report by Mundiñano, which could reflect a compensatory mechanism [47].

Fig. 1. Alterations of PD motor symptoms in the asymptomatic and symptomatic PD mice with MPTP/p injections. At the asymptomatic and symptomatic stages, motor behavior tests (A: open field test, B: rotarod test) were performed. Values are given as the mean ± SEM. ###p < 0.001 compared with the control group.

In conclusion, our results demonstrated the onset of not only premotor symptoms such as intestinal dysmotility and olfactory loss but also of movement deficits, depending on the number of MPTP/p injections. Our data also showed that there were changes in factors related to the premotor symptoms of early PD, including the number of dopaminergic, cholinergic, and microglial cells, and the production of nitric oxide and α-synuclein, following administration of MPTP/p. Because these features resemble the pathogenic conditions in patients in the early stages of PD, this animal model could potentially be used to study the early PD events accompanied by several non-motor symptoms and alterations in their regulatory factors.

Fig. 2. Alterations of dopaminergic neuronal damage and striatal DAT levels regulatory bioanalysis in the asymptomatic and symptomatic PD mice with MPTP/p injections. Immediately after the behavior test of the last group, brain tissues were obtained for immunohistochemical analysis (A: immunostaining of TH in SNpc, B: immunostaining of TH in ST, C: immunostaining of DAT in ST). Values are given as the mean ± SEM. #p < 0.05 and ###p < 0.001 compared with the control group.

Fig. 3. Alterations of PD non-motor symptoms on the asymptomatic and symptomatic mice with MPTP/p injections. At the asymptomatic and symptomatic stages, non-motor behavior tests (A: bead expulsion test, B: buried pellet test) were performed. Values are given as the mean ± SEM. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the control group.