Probenecid Mouse Model of Parkinson’s Disease

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NEUROSCIENCE 1

RESEARCH ARTICLE K. C. Biju et al. / Neuroscience xxx (2018) xxx–xxx

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Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson’s Disease

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K. C. Biju, a Robert C. Evans, a Kripa Shrestha, a Daniel C. B. Carlisle, a Jonathan Gelfond b and Robert A. Clark a,c*

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a

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Department of Epidemiology and Biostatistics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, United States

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c

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Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, United States

South Texas Veterans Health Care System, 7400 Merton Minter Blvd, San Antonio, TX 78229, United States

Abstract—Mitochondrial dysfunction and oxidative stress are very prominent and early features in Parkinson’s disease (PD) and in animal models of PD. Thus, antioxidant therapy for PD has been proposed, but in clinical trials such strategies have met with very limited success. Methylene blue (MB), a small-molecule synthetic heterocyclic organic compound that acts as a renewable electron cycler in the mitochondrial electron transport chain, manifesting robust antioxidant and cell energetics-enhancing properties, has recently been shown to have significant beneficial effects in reducing nigrostriatal dopaminergic loss and motor impairment in acute toxin models of PD. However, no studies have investigated the impact of this promising agent in chronic models or for olfactory dysfunction, an early non-motor feature of PD. To test the efficacy of low-dose MB for olfactory dysfunction, motor symptoms, and dopaminergic neurodegeneration, mice were injected with ten subcutaneous doses of 25 mg/kg MPTP, plus 250 mg/kg intraperitoneal probenecid or saline/probenecid at 3.5-day intervals. Following the onset of olfactory dysfunction, MPTP/probenecid (MPTP/p) and saline/probenecid mice were provided drinking water with or without 1 mg/kg/day MB. Oral delivery of low-dose MB significantly ameliorated MPTP/p-induced deficits in motor coordination, as well as degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH-positive terminals in the striatum. Importantly, olfactory dysfunction was ameliorated by MB treatment, whereas this benefit is not observed with currently available anti-Parkinsonian medications. These results indicate that low-dose MB is a promising neuroprotective intervention for both motor and non-motor features of PD. Published by Elsevier Ltd on behalf of IBRO. Key words: neurodegeneration, Parkinson’s disease, olfactory dysfunction, dopamine, nesting behavior, substantia nigra.

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INTRODUCTION

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Parkinson’s disease (PD) has long been viewed as a motor system disorder, promoting an era of therapeutics focused almost exclusively on dopamine neurons and motor symptoms. However, the disease is accompanied by numerous non-motor manifestations that add significantly to the overall level of disability (Marras and Chaudhuri, 2016). Current PD treatment, based primarily on pharmacological replacement of dopamine to treat motor symptoms, provides only symptomatic relief that

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typically wanes in efficacy after a few years. As therapeutic effectiveness diminishes, patients begin to suffer from drug-resistant motor symptoms (speech impairment, abnormal posture, gait and balance problems), as well as increasing drug side effects (psychosis, motor fluctuations, dyskinesia). Thus, replacing lost dopamine is clearly insufficient for arresting the disease progression. In fact, many non-motor symptoms (olfactory, sleep, and autonomic dysfunctions, anxiety, depression) precede the motor symptoms by years, or even decades (Pfeiffer, 2016; Schapira et al., 2017). Since non-motor symptoms are likely caused by the same protein aggregation pathologic mechanism that leads to motor symptoms, this temporal pattern begs the question of whether disease-modifying therapeutic strategies aimed at nonmotor symptoms during the prodromal stage might also prevent or delay dopaminergic neurodegeneration and motor symptoms, thereby providing much-needed therapeutic benefit.

*Correspondence to: R. A. Clark, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, United States. Fax: +1-210-567-4654. E-mail address: [email protected] (R. A. Clark). Abbreviations: 6-OHDA, 6-hydroxydopamine; L-DOPA, L-3,4dihydroxyphenylalanine; MB, methylene blue; MPP+, 1-methyl-4phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri dine; OD, optical density; p, probenecid; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; STRdl, dorsolateral region of striatum; STRv, ventral region of striatum; TH, tyrosine hydroxylase. https://doi.org/10.1016/j.neuroscience.2018.04.008 0306-4522/Published by Elsevier Ltd on behalf of IBRO. 1

Please cite this article in press as: Biju KC et al. Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson’s Disease. Neuroscience (2018), https://doi. org/10.1016/j.neuroscience.2018.04.008

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Methylene blue (MB) (methylthioninium chloride) is a small-molecule synthetic heterocyclic organic compound. At low levels (nanomolar), MB acts as a renewable electron cycler in the electron transport chain, thereby enhancing cytochrome oxidase activity and ATP production, promoting cell survival (Rojas et al., 2012). MB also decreases production of reactive oxygen species in the electron transport chain via bypassing complex I/III activity. Hence, MB has the potential to mitigate oxidative damage. Indeed, mitochondrial impairment and oxidative stress are very prominent features in the PD brain (Lavrovsky et al., 2000; Ahlskog, 2005). Tissue samples from human PD patients provide convincing evidence for oxidative damage to a broad range of macromolecules, such as nucleic acids, lipids, and proteins (Sanders and Greenamyre, 2013). Moreover, in vitro and in vivo studies suggest that oxidative stress can induce a-synuclein aggregation (Hashimoto et al., 1999; Kowall et al., 2000), an early event in the initiation of PD. Interestingly, the olfactory bulb, one of the two brain regions where a -synucleinopathy first appears, is also very susceptible to mitochondrial compromise, oxidative stress, and excitotoxicity. Furthermore, olfactory dysfunction is an early warning sign, with olfactory loss occurring in up to 90% of PD patients. Although the precise mechanisms of mitochondrial dysfunction and oxidative stress in the etiology or pathogenesis of PD are yet to be elucidated, available data suggest that they contribute significantly to neurodegeneration in PD (Sanders and Greenamyre, 2013). In addition to acting as a renewable electron cycler in the mitochondrial electron transport chain, MB exerts its effect through other mechanisms relevant to PD – e.g., inducing autophagy, promoting neurogenesis, elevating monoamine levels through inhibition of monoamine oxidases, inhibiting nitric oxide synthase and nitric oxide-sensitive soluble guanylate cyclase, and blunting inflammatory responses (Deutsch et al., 1996; Sontag et al., 2012; Guerrero-Munoz et al., 2014). Recently, MB has been shown to have significant beneficial effects in reducing nigrostriatal dopaminergic loss, motor impairment, and attentional deficits in acute toxin models of PD (Rojas et al., 2009; Wen et al., 2011; Smith et al., 2017). However, no studies have investigated the impact of this promising agent in chronic models or for olfactory dysfunction, an early non-motor feature of PD. Typically, non-motor features precede motor symptoms. To the extent that non-motor and motor deficits share similar mechanisms, drugs that mitigate non-motor symptoms (e.g., olfactory dysfunction) should have a better chance of being disease modifying than those that address only motor symptoms, provided the treatment is initiated at an early stage. Thus, olfactory efficacy may forecast the prevention or delay in onset of motor dysfunction in clinical trials of novel diseasemodifying drugs. Additionally, to the best of our knowledge, this is the first study on the effect of MB on a chronic MPTP/probenecid mouse model of PD. Given the diverse pathological processes and heterogeneity in the expression and progression of the clinical manifestations of PD, as well as the likelihood that there are multiple contributing etiologic factors, there is no single ideal animal

model. Therefore, testing of promising drug candidates in multiple models is critical for assessing predictive value of successful translation of drugs into the clinic.

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EXPERIMENTAL PROCEDURES

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Mice

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Male C57BL/6J mice at 10 weeks of age were grouphoused in a 14/10-h light/dark cycle. Animal husbandry was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, Society for Neuroscience Policies on the Use of Animals and Humans in Neuroscience Research, and institutional requirements for animal care. A total of 30 mice were used. Baseline general activity in the open field, as well as proper righting reflex, were assessed before the experiment. All treatments and behavioral tests were performed during the light cycle. For the behavioral tests the mice were acclimatized to the testing room for at least 1 h prior to the session. The smallest possible number of mice was used (based on power calculations) and all efforts were made to minimize suffering.

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MPTP/probenecid treatment

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One week after the baseline assessment of general activity and righting reflex, the mice in one group were treated with ten subcutaneous doses of 25 mg/kg MPTP (Sigma, St. Louis, MO, USA) in saline, plus 250 mg/kg intraperitoneal probenecid (Sigma) in Tris–HCl buffer at 3.5-day intervals for a total of five weeks (MPTP/p group; n = 20; Fig. 1) (Meredith et al., 2008). Mice in the other group received saline and probenecid on the same schedule and were used as controls (Saline/p group; n = 10).

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MB treatment

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To assess the therapeutic effects of MB in early-stage disease, eleven days after the first dose of MPTP/p the animals were screened for olfactory dysfunction by recording the time spent in familiar versus unfamiliar compartments (Prediger et al., 2010). Following the onset of olfactory dysfunction (day 11 post MPTP/p), one subgroup from each of the MPTP/p (n = 10) and Saline/p (n = 5) mice were given 1 mg/kg/day MB, USP (Akorn, Lake Forest, IL, USA) in drinking water (Hosokawa et al., 2012), whereas the remaining MPTP/p (n = 10) and Saline/p (n = 5) mice were given regular drinking water (Fig. 1). Of the 20 mice in the MPTP/p arm 5 died of acute toxicity during MPTP/p treatment, thus reducing the total number in the MPTP/p arm to 15 (7 MPTP/p and 8 MPTP/p+ MB, as shown in Fig. 2). Body weight and liquid consumption were monitored throughout the course of MB treatment. Water intake averaged between 4.3 and 4.7 ml/30 g body weight/day. MB did not induce any significant change in liquid consumption. The selected concentration of MB in the drinking water was based on the assumption that a 30-g mouse drinking 4.5 ml of liquid/day would receive MB at 1 mg/kg body

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mice were allowed to grasp the center point of a 6-mm diameter copper bar (38 cm long, held 49 cm above the floor) with their forepaws only and the time to falling from the bar was recorded, or alternatively reaching one end of the bar within the maximum test time of 30 s Fig. 1. Time course of MPTP/p and MB treatments, as well as performance of behavioral tests. (completion of the task) was noted (Deacon, 2013). Scoring of the horizontal bar test results was as follows: falling between 1–5 s = 1; falling between 6–10 s = 2; falling between 11–20 s = 3; falling between 21–30 s = 4; falling after 30 s = 5; completing the task = 5. Test for general anosmia: General anosmia was assessed using the buried food retrieval paradigm (Lehmkuhl et al., 2014). The mice were placed in a testing cage in which a sweetened cereal pellet (Cap’n Crunch; Quaker Oats Company) was buried 0.5 cm below the bedding so that it was not visible. The testing cage consisted of a clean mouse cage that was filled 3 cm with fresh bedding. The retrieval time was recorded from the instant the mouse was released in the center of the testing cage until the cereal pellet was found (maximum 15 min). Retrieval time for an unscented glass marble and time to find an exposed (vs. hidden) cereal pellet were Fig. 2. MPTP/p treatment induced olfactory dysfunction. MPTP/p (n determined similarly to assess any potential confounding = 7) and MPTP/p+ pre-MB (n = 8) mice showed no preference for of the buried cereal test by deficits in motor coordination the familiar compartment when tested 11 days after the first injection of MPTP/p, whereas Saline/p (n = 5) and Saline/p+ pre-MB (n = 5) or anxiety-related digging behaviors. Similarly, since food mice spent significantly more time in the familiar compartment. Data was used as a cue, body weight and food intake were are expressed as mean ± S.E.M of the time (seconds) spent in each monitored prior to the test to assess any potential concompartment. Number of animals used (n) in each treatment group founding of the buried cereal test by differences in food are the same for Figs. 2–8. intake among the treatment groups. Olfactory discrimination tests: The mice were tested weight/day. MB treatment continued till the end of the for their ability to discriminate between two palatable experiment. odors using a habituation/dishabituation paradigm as described before (Lehmkuhl et al., 2014). Briefly, a scented (almond flavor) cartridge was presented in six Behavior tests consecutive trials for a duration of two minutes with Open field: The open-field test was performed as inter-trial interval being one minute. Habituation was described before (Dunnett et al., 1998). Briefly, mice were demonstrated by a gradual decrease in sniffing toward acclimatized to the testing room for at least 1 h prior to the repeated presentation of the same odor (here almond each test, which was performed between 12 PM and 5 flavor), whereas dishabituation was demonstrated by a PM with an illumination level of 321.09 lux from a reinstatement of sniffing with the presentation of a novel recessed fluorescent ceiling light. The floor of the white odor (here banana flavor) in the seventh trial. To assess 60
60-cm open field was subdivided by blue lines into the onset of olfactory dysfunction after MPTP/p treatment, 25 12
12-cm squares. For each test, mice were placed we used a paradigm based on the time spent in familiar individually into the open field, and their behavior was versus unfamiliar compartments (Prediger et al., 2010). videotaped over 10 min. Between every two tests the This task is based on the fact that rodents normally disopen field was wiped with ethanol solution and dried to close preference for places impregnated with their own remove odor trails. Locomotor activity was defined as odor (familiar compartment). Thus, mice with intact olfacthe number of squares entered in 10 min. tion will spend more time in a familiar compartment when Static bar test: The test was performed as described given a choice between familiar vs. unfamiliar compartbefore (Deacon, 2013). Briefly, a 9-mm diameter wooden ments. In this test, each mouse was placed in a cage split rod (60 cm long) was fixed by a G-clamp to a laboratory into two equal areas separated by an open door. The shelf such that the rod protrudes horizontally. The mouse mouse could choose between a compartment with sawwas placed at the distal end of the rod facing outward and dust that the same mouse had occupied for three days performance metrics were recorded for: (i) time to reorient before the test (familiar compartment), and a compart180 degrees from the starting position (T-turn), and (ii) ment with fresh sawdust (unfamiliar compartment). The time to travel to the shelf end (T-travel). time spent in each compartment in a 5-min trial was Horizontal bar test: The horizontal bar test measures recorded. both motor coordination and strength. In this test the Please cite this article in press as: Biju KC et al. Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson’s Disease. Neuroscience (2018), https://doi. org/10.1016/j.neuroscience.2018.04.008

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Nesting behavior: Nest building was assessed as described previously (Deacon, 2006). Briefly, the mice were individually housed in a clean plastic cage with approximately 2 cm of bedding lining the floor. One hour prior to the onset of the dark phase of the lighting cycle, a piece of pre-weighted 51-mm-square
5-mm-thick NestletTM cotton pad (NestletsTM, Ancare Corp., Belmont, New York) was placed on the floor of each cage. The nests were assessed 24 h later on a rating scale of 1–5 (Deacon, 2006) and the weight of any untorn (>0.1 g) Nestlet piece was recorded.

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Tissue processing

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The mice were anesthetized with an overdose of ketamine HCl/xylazine HCl solution (Henry Schein Animal Health, Dublin, OH, USA) and perfused transcardially with 10–20-ml ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by an equal volume of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed overnight in the same fixative at 4 °C. The tissues were cryoprotected in sequential solutions of sucrose (10% for 2 h, 20% for 2 h, and 30% for 24–48 h), and then embedded in Tissue-Tek OCT compound. Brain sections were prepared at 30 lm thickness in the coronal plane using a Leica CM 1950 cryostat. Four series of slides, each containing every fourth section, were prepared for substantia nigra, and six series of every sixth section were prepared for striatum. Anatomical landmarks were determined according to Paxinos and Franklin (2001).

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Immunohistochemistry

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Brain sections were immunostained using the standard avidin–biotin-complex (ABC) method. Briefly, sections were treated with 1% bovine serum albumin in PBS containing 0.3% Triton X-100 for 30 min and then incubated with rabbit anti-tyrosine hydroxylase (TH; EMD Millipore, Temecula, CA, USA) at 1:2000 dilution for 48 h at 4 °C. Sections were rinsed in PBS and incubated with biotinylated goat anti-rabbit secondary antibody (1:200) for 1 h at room temperature. After another rinse in PBS, the sections were incubated with avidin–biotin peroxidase complex (ABC-Elite Kit, Vector Laboratories, Burlingame, CA) at room temperature for 1 h. The chromogen used was either 3-amino-9-ethyl carbazole (AEC Kit, Vector Laboratories) or 3,30 diaminobenzidine tetrahydrochloride (ImmPACT DAB EqV Kit, Vector Laboratories). Slides containing the brain sections were coverslipped and images were analyzed using a regular light microscope (Nikon Eclipse TE2000-U). Stringent control procedures were utilized to ensure specificity of immunoreactions.

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Quantification of TH-positive and Nissl-stained neurons Total number of TH-positive neurons in substantia nigra pars compacta (SNpc) was estimated by counting the number of neurons from every fourth serial section throughout the entire extent of the SNpc using a 40x

objective. The slides were blind-coded and neurons showing a clear TH-positive cytoplasm around non-stained nuclei were counted. The number of Nissl-stained neurons in the SNpc was similarly estimated. To make sure that cell counts were not affected by cell size and/or tissue shrinkage due to treatment, the pixel areas of substantia nigra and Nissl-stained cells were estimated from digitized images of midbrain sections using ImageJ software. Using this method we found no significant changes in cell size or tissue shrinkage among the treatment groups.

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Optical density

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Optical densities (OD) of the TH-positive fibers in the striatum were measured from digitized images of every sixth section using NIH ImageJ software. The measurements were taken from dorsolateral aspects of the striatum since they receive the majority of innervation from SNpc dopaminergic neurons. The relative OD of TH-positive fibers in the striatum was calculated by subtracting the background OD from the measured OD of the dorsolateral aspects of the striatum (Biju et al., 2010). Conditions for tissue processing, immunostaining, and image acquisition were kept constant for all animals.

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1-Methyl-4-phenylpyridinium (MPP+) assay

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Striatal levels of MPP+ were estimated from a separate group of male C57BL/6J mice. The mice (n = 12) were treated with three subcutaneous doses of 25 mg/kg MPTP (Sigma, St. Louis, MO, USA) in saline, plus 250 mg/kg intraperitoneal probenecid (Sigma) in Tris–HCl buffer at 3.5-day intervals (Meredith et al., 2008). Half of these mice (n = 6) were given 1 mg/kg/day MB, USP (Akorn, Lake Forest, IL, USA) in drinking water (Hosokawa et al., 2012), starting from one day prior to MPTP/p treatment and continuing till the end of the experiment (MPTP/p+ MB group). The remaining MPTP/p mice (n = 6) were given regular drinking water. A third group of mice (n = 5) were used as untreated controls. Body weights and liquid consumption were monitored throughout the duration of the experiment. Mice were killed 4 h after the third injection of MPTP/p. The brains were quickly dissected out, placed in a mouse brain slicer matrix (brain blocker) and then chilled on dry ice for 30 s. Striatum was quickly dissected out from 1-mm-thick coronal sections of the forebrain, placed into pre-weighed tubes, snap-frozen on dry ice and then stored at 80 °C until analysis. The striatum was homogenized by sonication in 9 parts (w/v) of cold 5% trichloroacetic acid and the homogenates were centrifuged at 15,000g for 20 min at 4 °C (Jackson-Lewis and Przedborski, 2007). The supernatants were analyzed for MPP+ levels using a Bio Rad SmartSpec Plus spectrophotometer at 295 nm (UV detection). A standard curve was generated using MPP + iodide (Sigma, Cat # D048). The absorbance of untreated brain supernatant (background) was deducted from the absorbance of MPTP/p-treated mice.

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Statistical analysis

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We used a two-way ANOVA or a nonparametric equivalent to assess the effects of MPTP/p and MB on

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all measures. We tested the ANOVA assumption of equality of variances with Levine’s test, and if there was significant (P  0.05) heteroscedasticity, we used the log transformation. We used Tukey’s correction in ANOVA for all pairwise comparisons between groups to account for multiple testing. Some measures did not satisfy normality because the Shapiro test was significant or due to measures having a restricted range (e.g., the horizontal bar score taking integer values from 1 to 5). For these non-Gaussian measures, we used pairwise Wilcoxon rank sum tests with the Bonferroni correction of P-values (Pcorrected = 6Praw). We assessed olfactory dysfunction using a paired t-test to compare time spent in familiar and unfamiliar compartments within each group, and we used an unpaired t-test for comparing means of MPP+ levels in the brain. Results were expressed as mean ± S.E.M. and differences considered significant at P  0.05. Tukey’s studentized range statistics and t-statistics were reported as t(df) and Wilcoxon statistics were reported as W(n1, n2). Statistical analyses were performed in R version 3+ (Vienna, Austria) using an accountable data analysis process.

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RESULTS

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Olfactory dysfunction is an early warning sign of PD. Therefore, to assess the therapeutic effects of MB in early-stage disease and provide a close parallel to early PD management in patients, eleven days after the first dose of MPTP/p the animals were screened for the onset of olfactory dysfunction by recording the time spent in familiar versus unfamiliar compartments (since mice with intact olfaction will normally choose to spend more time in a ‘‘familiar compartment”, an environment impregnated with their odor). We used this paradigm over more traditional buried food retrieval paradigm during MPTP/p treatment to avoid any additional stress associated with overnight fasting in the buried food retrieval paradigm. By eleven days after the first dose of MPTP/p treatment the animals had developed olfactory dysfunction, as indicated by the equal amounts of time spent in familiar and non-familiar compartments (lack of preference for familiar compartment; t(6) = 0.12, P  0.9 for MPTP/p and t(7) = 0.41, P  0.7 for MPTP/p+ Pre-MB; Fig. 2). Following the onset of olfactory dysfunction, the mice were given 1 mg/kg/day MB in drinking water or regular drinking water, as described in Experimental Procedures. To avoid any potential influence on behavior of peripheral toxicity associated with MPTP intoxication, all subsequent behavioral tests were performed at least 15 days after the final dose of MPTP/p (Fig. 1).

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Effects of orally delivered MB on deficits in motor coordination At day 41 after the start of MB treatment (i.e., day 16 after the last dose of MPTP/p), the animals were tested for motor coordination. MPTP/p treatment resulted in significant impairment in motor performance assessed by the static rod test, as the MPTP/p mice took

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significantly more time to turn (log10 transformed, t(21) = 4.37, P  0.001) and traverse (log10 transformed, t( 21) = 6.06, P  0.001) the rod (Fig. 3A, B). Orally delivered MB significantly improved these MPTP/pinduced deficits in motor performance, seen as decreased time to turn (t(21) = 4.16, P  0.002) and traverse (t(21) = 5.63, P  0.001) the rod (Fig. 3A, B). To confirm the positive effect of MB on MPTP/p-induced deficits in motor performance, one day after the static bar test, we performed the horizontal bar test, which measures both motor coordination and strength. The MPTP/p-treated animals scored poorly, compared with

Fig. 3. Orally delivered MB improved MPTP/p-induced impairment in motor coordination and strength. (A, B) Results of static bar test (mean ± S.E.M). MPTP/p group mice (n = 7) took significantly more time to turn (A) and traverse (B) a 9-mm rod, whereas the times MPTP/p+ MB group (n = 8) took to turn (A) and traverse (B) the rods were closer to those of Saline/p (n = 5) and Saline/p+ MB (n = 5) control mice. (C) Result of horizontal bar test (mean ± S.E.M.). MPTP/p treatment decreased scores for latency to fall from a 6-mm horizontal bar, whereas the scores of the MPTP/p+ MB group were restored to those of Saline/p and Saline/p+ MB control groups.

Please cite this article in press as: Biju KC et al. Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson’s Disease. Neuroscience (2018), https://doi. org/10.1016/j.neuroscience.2018.04.008

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those in the Saline/p group (Fig. 3C; W(5, 7) = 1.5, P  0.05). However, the MPTP/p+ MB mice performed similarly in the horizontal bar test to those in the Saline/ p group (Fig. 3C; W(5, 8) = 21.5, P  1), confirming that MB ameliorated MPTP/p-induced impairment in motor coordination. Effects of orally delivered MB on olfactory dysfunction Mice displayed olfactory dysfunction as early as eleven days after the first dose of MPTP/p treatment. However, to avoid confounding by MPTP/p intoxication-related declines in activity, we first assessed general activity in the open-field test on day 24 after the last dose of MPTP/p. Overall activity levels were similar in MPTP/p and Saline/p control groups (Fig. 4; t(21) = 0.02, P  1). Twenty days later, the mice were tested for olfactory dysfunction (general anosmia) using the buried food test paradigm. MPTP/p mice took more time than control mice (Saline/p) to retrieve the hidden cereal (Fig. 5A; log10 transformed, t(21) = 5.17, P  0.001). However, oral delivery of MB resulted in mitigation of olfactory dysfunction, as the retrieval times for MPTP/p+ MB mice were significantly shorter, compared with the MPTP/p group (Fig. 5A; t(21) = 3.01, P  0.03). Since the retrieval time for an unscented glass marble or time to find an exposed (vs. hidden) piece of cereal was not significantly different between the treatment groups (data not shown), it is unlikely that the results of the buried cereal test were confounded by motor coordination deficits or anxiety-related digging behaviors. Body weight and food intake were monitored prior to the general anosmia test, since food was used as a cue in the test. Although slight weight loss was observed in the MPTP/p groups immediately after the MPTP/p treatment, the animals had regained weight by the time of the test for general anosmia (50 days-post last dose of MPTP/p), and body weights were not significantly different among the four treatment groups (Fig. 5B; for example, t(21) = 1.01, P  0.75 for MPTP/ p vs. MPTP/p+ MB). The food intake profiles of the four treatment groups were also similar (data not

Fig. 4. General activity in the open-field test performed on day 24 after the final dose of MPTP/p. Overall activity levels (mean ± S.E.M. of the number of squares entered in 10 min) were similar among all the groups tested (P  1).

Fig. 5. Oral delivery of MB restored MPTP/p-induced anosmia (A) and deficit in olfactory discrimination (C). (A) Histogram plots of the retrieval time (mean ± S.E.M.) of a hidden cereal in buried food pellet retrieval test for general anosmia. (B) Histogram plots of body weight measured one day prior to test for anosmia. (C) Plots of sniffing time versus odor trial number for mice habituated to the first odorant (almond flavor, 1:100 dilution) by repeated presentation of the odor at 1-min intervals. On the seventh trial, the novel odor (banana flavor, 1:100 dilution) was presented to test for discrimination following habituation to the first odorant. Oral delivery of MB prevented the MPTP/p-induced deficit in odor discrimination, as the MPTP/p+ MB group spent significantly more time sniffing the odor cartridge in trial seven, thereby resembling the Saline/p mice.

shown). Thus, it is unlikely that the results of the buried cereal test were confounded by any difference in foraging activity among the treatment groups. In addition to deficits in odor detection, up to 90% of PD patients exhibit impairment in odor identification and discrimination. Therefore, one week after the buried food test we assessed the mice for their ability to discriminate between two palatable odors using a

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habituation-dishabituation paradigm. As shown in Fig. 5C, oral delivery of MB mitigated the MPTP/p-induced deficit in odor discrimination, as the MPTP/p+ MB group spent significantly (t(21) = 3.24, P  0.02) more time sniffing the odor cartridge in trial seven, thereby resembling the Saline/p mice. Of note, the Saline/p+ MB control group outperformed all other treatment groups, as indicated by a pronounced decline in sniffing toward the repeated presentation of the first odor (Fig. 5C).

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Nest building is a motivated, goal-directed behavior requiring orofacial and forelimb motor coordination. Deficits in nesting behavior correlate with loss of striatal dopamine, as seen in PD. Nest building behavior can thus be used effectively for assessing efficacy of antiParkinsonian therapeutics. We observed consistent deficits in nest building in the MPTP/p group from very early in the course of MPTP/p treatment. However, we did not score nesting behavior during MPTP/p treatment because the mice were group housed to minimize any associated distress. Thirty-seven days after the final dose of MPTP/p, the mice were single-housed temporarily and their nesting behavior was assessed using Deacon’s five-point rating system, as well as measuring the weight of any unshredded portion of the Nestlet. The Saline/p and Saline/p+ MB control group built their nests nearly perfectly (score: 4.8 ± 0.2; Fig. 6A, B). However, as shown in Fig. 6A, B, MPTP/ptreated mice exhibited severely impaired nesting behavior (score: 1.57 ± 0.2) with large pieces of Nestlet left untorn (Fig. 6C), whereas the MPTP/p+ MB group demonstrated significant improvement in nest-building behavior (score: 3.5 ± 0.189; W(7, 8) = 56, P  0.01) with significantly smaller pieces of the Nestlet left untorn (Fig. 6C; t(21) = 4.57, P  0.001).

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Effects of orally delivered MB on degeneration of the nigrostriatal dopaminergic system Following the behavioral tests, at 63 days after the final dose of MPTP/p, animals were killed and quantitative analysis of TH-positive and Nissl-stained neurons in the substantia nigra pars compacta (SNpc), as well as the density of TH-positive terminals in the striatum were performed to assess the potential neuroprotective effects of MB on the nigrostriatal dopaminergic system. The organization and intensity of TH immunoreactivity were essentially similar in the Saline/p and Saline/p+ MB groups (Fig. 7A). Importantly, up to 66% of TH-positive neurons in the SNpc were lost in MPTP/p mice, compared with the Saline/p mice (Fig. 7B; t(21) = 9.57, P  0.001). Nissl staining revealed similar loss of neurons in the SNpc of MPTP/p mice (Fig. 7B; t(21) = 16.41, P  0.001), indicating that the observed reduction in TH-positive neurons in the SNpc was indeed due to neurodegeneration, rather than potential TH downregulation in the absence of neurodegeneration. The TH-positive dendritic fiber networks in the substantia nigra pars reticulata (SNpr)

of MPTP/p mice were also dramatically reduced (Fig. 7A). In contrast, only about 28% of TH-positive neurons in the SNpc were lost in the MPTP/p+ MB mice (t(21) = 4.21, P  0.002). Moreover, the density of the SNpr TH-positive dendritic fiber network was largely preserved in MPTP/p+ MB mice, relative to Saline/p or Saline/p+ MB mice. Similar results were observed for dopamine fiber terminals in the dorsal region of the striatum (Fig. 8A). Since the dorsolateral aspects of the striatum receive the largest share of innervation from dopamine neurons of the SNpc, OD measurements were performed on these aspects to quantify the intensity of TH staining. TH immunoreactivity within the dorsolateral striatum was similar in Saline/p and Saline/ p+ MB mice (Fig. 8B). Relative to the Saline/p controls, there was an average 67% loss of TH staining intensity in MPTP/p mice (t(21) = 11.6, P  0.001), whereas the loss in MPTP/p+ MB mice was only 28% (Fig. 8B; t (21) = 4.81, P  0.001). At day nine after the start of MB treatment (4h postthird dose of MPTP/p) striatal levels of MPP+ from a separate cohort of mice treated with MPTP/p or MPTP/p + MB (see Experimental Procedures for details) were analyzed for potential interaction between MB and MPTP metabolism. In MPTP/p mice, the mean striatal MPP+ level was 7.93 ± 1.19 ng/mg tissue. At 7.45 ± 1 .03 ng/mg tissue, the level of MPP+ in MPTP/p+ MB mice was similar to that in MPTP/p mice (Fig. 8C). Similar concentrations of MPP+ have been reported when mouse striatal extracts were analyzed 3 h post 30 mg/kg subcutaneous injections of MPTP (Hows et al., 2004). Our results indicate that with an oral dose of 1 mg/kg/day, MB does not have a significant effect (t(10) = 0.308, P  0.6) on the conversion of MPTP to its active metabolite MPP+ by monoamine oxidase B (MAO B).

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We demonstrated here that daily low-dose oral delivery of MB ameliorated deficits in olfaction and motor coordination, as well as nigrostriatal dopaminergic degeneration in a chronic MPTP/p model of PD. To the best of our knowledge, this is the first demonstration of a drug candidate with a therapeutic impact on both olfactory dysfunction and motor features of the disease. Mitochondrial impairment and oxidative stress are very prominent features in the PD brain (Lavrovsky et al., 2000; Ahlskog, 2005). Tissue samples from human PD patients provide evidence for oxidative damage to many categories of macromolecules (Sanders and Greenamyre, 2013), and in vitro and in vivo studies suggest that oxidative stress can induce a-synuclein aggregation (Hashimoto et al., 1999; Kowall et al., 2000). Thus, antioxidant therapy for PD has been proposed. Antioxidants such as coenzyme Q10, creatine, vitamin E, vitamin C, b-carotene, urate, and apocynin, as well as the modified forms of some of these antioxidants that selectively target mitochondria – MitoQ, MitoVitE, MitoTEMPOL, SkQ1, MitoApocynin, SS-20, and SS-31 – have shown promising neuroprotective effects in some PD models (Jin et al., 2014). However, these strategies have

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Fig. 6. MB treatment ameliorated deficits in nesting. (A) Representative images of nest construction. Saline/p and Saline/p+ MB groups completely shredded the Nestlet and arranged the material into a well-defined nest, whereas the MPTP/p group only partially shredded the Nestlet. MPTP/p+ MB group mostly shredded the Nestlet and arranged the material into a nest. (B, C) Plots of quantitative data showing the results from the nest construction experiment. Deacon’s nesting scoring scale: 1 = Nestlet not noticeably touched; 2 = Nestlet partially shredded; 3 = Nestlet mostly shredded but no identifiable nest site; 4 = an identifiable but flat nest; 5 = a perfect nest with wall surrounding mouse body.

Fig. 7. Orally delivered MB protected nigral dopaminergic neurons from MPTP/p-induced degeneration. (A) Midbrain sections showing THimmunostained cell bodies and their processes in the substantia nigra. Whereas MPTP/p treatment dramatically reduced the number of TH-positive cell bodies and fibers in the substantia nigra pars compacta (SNpc) of MPTP/p group, a significant protective effect was observed in the MPTP/p+ MB group. (B) Plots of number (mean ± S.E.M.) of TH-positive and Nissl-stained neurons in the SNpc. SNpr, substantia nigra pars reticulata.

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had very limited success in clinical trials (Sanders and Greenamyre, 2013). Although multiple factors likely contribute to the failure of such trials, a key issue is the mode of action of these antioxidants, all of which work by scavenging free radicals. Thus, such agents must be delivered

to the target site at the critical time before the reactive free radicals can oxidize macromolecules, a challenging requirement for a chronic condition like PD. In contrast, by acting as an electron coupler in the mitochondrial electron transport chain, MB preempts oxidant injury by

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Fig. 8. Orally delivered MB protected dopaminergic terminals in the striatum from MPTP/p-induced degeneration. (A) Coronal sections of forebrain showing immunostaining of TH-positive terminals in the striatum. In the MPTP/p group, there was a dramatic reduction in the density of TH-positive terminals in the dorsolateral region of the striatum (STRdl), whereas a substantial portion of TH-positive terminals was spared in the striatum of MPTP/p+ MB mice. Note that the TH-positive terminals in the ventral region of the striatum (STRv) were largely preserved in the MPTP/p group. (B) Plots of quantitative data illustrating a protective effect of MB treatment as assessed by optical density measurement of TH-positive terminals in the STRdl. (C) Plots of quantitative data showing striatal concentrations of MPP+ in a separate cohorts of MPTP/p (n = 6) and MPTP/p+ MB (n = 6) mice.

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reducing the generation of free radicals. Indeed, MB has significant beneficial effects in reducing nigrostriatal dopaminergic loss and motor impairment in acute toxin models of PD, such as the rat rotenone model (Rojas et al., 2009; Wen et al., 2011) and rat 6-hydroxydopamine (6-OHDA) model (Smith et al., 2017). Our demonstration of therapeutic efficacy of low-dose MB for motor coordination and nigrostriatal dopaminergic loss in the chronic MPTP/p mouse model align with the studies in rat toxin models (Rojas et al., 2009; Wen et al., 2011; Smith et al., 2017), while providing novel evidence for mitigation of olfactory dysfunction. Notably, olfactory dysfunction is an early warning sign of PD, with olfactory loss occurring in up to 90% of PD patients. The olfactory bulb is also one of two brain regions where a-synucleinopathy first appears. However, the pathogenesis of this olfactory loss in PD remains poorly understood (Doty, 2012). Thus, further studies are needed to elucidate the precise mechanism of the MB effect on MPTP/p-induced olfactory dysfunction that we demonstrated. The relatively sparse myelination of the projection neurons in the olfactory system may

place a heavy metabolic burden on these neurons, making them easier targets for oxidative stressors associated with neurodegeneration (Braak and Del, 2009). The high metabolic activity and reduced antioxidant capacity of neurons of the microglia-rich olfactory bulb may render them easily susceptible to mitochondrial impairment, oxidative stress, and excitotoxicity (Doty, 2012). Thus, MB’s salutary effect on olfactory dysfunction in the current study is likely related to its action as a renewable electron cycler in the mitochondrial electron transport chain, resulting in decreased oxidative injury. In spite of the reported association between impaired olfaction and subsequent development of PD (Ross et al., 2008), the nature of the link between olfactory loss and dopaminergic neurodegeneration remains unknown. Since MB treatment mitigated both olfactory dysfunction and dopamine cell loss, the MPTP/p+ MB model may be useful as a discovery tool to study the link between olfactory loss and dopaminergic neurodegeneration. The fact that olfactory dysfunction is unaffected by currently available dopaminergic medications probably reflects the lack of involvement of the dopaminergic system in PD-related olfactory dysfunc-

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tion (Schapira et al., 2017). It is possible that both olfactory dysfunction and dopaminergic neurodegeneration share a common mechanism, such as mitochondrial dysfunction leading to oxidative stress. Since olfactory dysfunction antedates motor symptoms by years or even decades, development of drugs that successfully prevent olfactory dysfunction in PD could be highly rewarding in clinical trials, since olfactory efficacy might forecast neuroprotection against motor dysfunction. Since deficits in nesting behavior correlate with loss of striatal dopamine (Sager et al., 2010), as seen in PD, nesting behavior can reflect the efficacy of antiParkinsonian therapeutics. In our study, nest-building deficits were observed in mice starting from 24 h after MPTP/ p intoxication and continuing until 39 days after the final dose. MB treatment resulted in significant improvement in nest-building behavior. Interestingly, L-3,4-dihydroxyphenylalanine (L-DOPA) has no significant effect on nest building deficits, despite improvements in motor control in MPTP-treated mice (Sager et al., 2010). Since nest building is a time-consuming task, it may be that the duration of L-DOPA-induced increase in striatal dopamine is too short to rescue nesting behavior. Alternately, it may be that nesting behavior, an executive function, depends not only on striatal dopamine, but also on an intact nigrostriatal dopaminergic system. The improvement in nesting behavior observed in our study was indeed accompanied by preservation of nigrostriatal dopaminergic neurons in the MPTP/p+ MB group of mice. Once inside the brain, MPTP is metabolized to its active form, MPP+ by the enzyme monoamine oxidase B (MAO-B). MB is a potent inhibitor of monoamine oxidase A (MAO A) and partial inhibitor of MAO B (Ramsay et al., 2007). Since MB and MPTP treatment were partially overlapping in our experiment, we assessed striatal levels of MPP+ to discern whether MB interfered with MPTP metabolism. Striatal concentrations of MPP+ in our study were close to those reported for mice on a similar MPTP dose (Hows et al., 2004) and no significant difference in MPP+ concentration was observed between MPTP/p and MPTP/p+ MB mice, indicating that MB was directly neuroprotective, rather than indirectly protective through its inhibitory action on MAO-B. Our data are consistent with literature indicating that the dose of MB used in our protocol was not high enough to cause a significant reduction in brain MPP+ levels. The halfmaximal inhibitory concentration (IC50) of MB for MAO A is about 164 nM, whereas that for MAO B is about 33 times higher at 5.5 lM (Ramsay et al., 2007). Data on the organ distribution of MB in rats indicate that intraperitoneal injection of 1 mg/kg MB, the same dose we used, leads to approximately 0.5 lM MB in the brain (Callaway et al., 2004) – i.e., about one tenth the IC50 value for MAO B inhibition. However, this same concentration was very effective in improving mitochondrial respiration (Callaway et al., 2004). Following its entry into mitochondria, MPP+ inhibits complex I in the electron transport chain, leading to ATP depletion, increased ROS production, and consequent activation of programmed cell death machinery. MB has high bioavailability to mitochondria, where it could act as

an electron coupler to bypass the MPP+-induced blockade of electron flow from complex I to IV and thereby minimize oxidative damage. Although the antioxidant properties of MB have been suggested to play a major role in its neuroprotective effect at low doses (Rojas et al., 2012), the therapeutic effect of MB on motor, non-motor, and pathological features reported in this study cannot be explained solely through an antioxidant effect. It has recently been shown that complex I is not required for MPP+ toxicity; instead there might be a complex I-independent mechanism intrinsic to dopaminergic neurons that renders them susceptible to MPP+ (Choi et al., 2008). MPP+ can increase the levels of both iron and a-synuclein in midbrain dopaminergic neurons (Mandel et al., 2004). In the presence of elevated iron levels, a-synuclein forms toxic aggregates (Mandel et al., 2004), leading to oxidative stress. Since MB has an inhibitory action on abnormal protein aggregation (Sontag et al., 2012; Guerrero-Munoz et al., 2014), it may either prevent MPP+-induced a-synuclein aggregation or abrogate oxidative stress induced by a-synuclein aggregates. Other mechanisms proposed for MPP+ toxicity are microtubule depolymerization and inhibition of glycolysis (Mazzio et al., 2003; Cappelletti et al., 2005; Choi et al., 2008). Although the effects of MB on microtubule depolymerization are not clear, it does greatly increase glycolysis in bovine articular cartilage chondrocytes under anoxic condition (Lee and Urban, 2002). Unlike traditional antioxidants, MB also exerts its effects through mechanisms relevant to PD, such as inducing autophagy, promoting neurogenesis, elevating monoamine levels through its inhibitory action on monoamine oxidases, inhibiting nitric oxide synthase and nitric oxide-sensitive soluble guanylate cyclase, and suppressing inflammatory responses (Deutsch et al., 1996; Sontag et al., 2012; Guerrero-Munoz et al., 2014). In short, elucidating the precise mechanisms of MPTP toxicity and determining which of these mechanisms is targeted by MB remain as objectives for further study. Given the diverse pathological processes and heterogeneity in the expression and progression of the clinical features of PD, no single drug candidate, here MB, is likely to provide a disease-modifying impact. Since motor symptoms start to appear only after 70– 80% of striatal DA and about half of nigral DA neurons have been lost (Bernheimer et al., 1973; McGeer et al., 1988; Fearnley and Lees, 1991), combining MB with traditional dopamine replacement therapies might enhance efficacy for both motor and non-motor symptoms. The efficacy of currently available dopamine replacement therapies for motor symptoms typically wanes after a few years and patients begin to suffer from drug-resistant motor symptoms, as well as increasing drug side effects. Additionally, L-DOPA can increase the rate of 6-OHDA generation and thereby add to oxidative stress (Maharaj et al., 2005). The potent antioxidant properties of MB could negate such toxicity when co-administered with LDOPA, thereby potentiating the effectiveness and extending the L-DOPA honeymoon period. Mitochondrial dysfunction and complex I inhibition have been found in both in the brain and peripheral tissues of patients with

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PD (Sanders and Greenamyre, 2013). The potent antioxidant and cell respiration-enhancing properties of MB, along with its other effects on autophagy and neurogenesis, as well as inhibitory action on monoamine oxidases, nitric oxide synthase, guanylate cyclase, and inflammatory responses, might thus provide a favorable multisystem therapeutic impact on both central and peripheral features of PD.

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This work was supported by a grant from the William and Ella Owens Medical Research Foundation and a Merit Review Grant from the Veterans Health Administration (1 101 BX 003157) awarded to Robert A. Clark. Biostatistical support was provided through a core resource of the San Antonio Older Americans Independence Center (NIH grant P30 AG044271). We thank Brian Hernandez for his assistance with the statistical analyses and William Friedrichs for help in measuring MPP+ levels in brain tissue.

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(Received 17 November 2017, Accepted 9 April 2018) (Available online xxxx)

Please cite this article in press as: Biju KC et al. Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson’s Disease. Neuroscience (2018), https:// doi.org/10.1016/j.neuroscience.2018.04.008

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