Neutralization of RANTES and Eotaxin Prevents the Loss of Dopaminergic Neurons in a Mouse Model of Parkinson Disease*

Parkinson disease (PD) is second only to Alzheimer disease as the most common human neurodegenerative disorder. Despite intense investigation, no interdictive therapy is available for PD. Recent studies indicate that both innate and adaptive immune processes are active in PD. Accordingly, we found a rapid increase in RANTES (regulated on activation normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in vivo in the substantia nigra pars compacta and the serum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. RANTES and eotaxin were also up-regulated in the substantia nigra pars compacta of post-mortem PD brains as compared with age-matched controls. Therefore, we investigated whether neutralization of RANTES and eotaxin could protect against nigrostriatal degeneration in MPTP-intoxicated mice. Interestingly, after peripheral administration, functional blocking antibodies against RANTES and eotaxin reduced the infiltration of CD4+ and CD8+ T cells into the nigra, attenuated nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Therefore, we conclude that attenuation of the chemokine-dependent adaptive immune response may be of therapeutic benefit for PD patients.

Although the etiology is poorly understood, PD is regulated by the adaptive arm of the immune system, and a number of recent studies have shown the involvement of inflammatory T cells in nigrostriatal degeneration (4 -8). In a normal adult brain, the cross-talk between the peripheral immune system and the brain is transient, and there is no evidence that it leads to the brain inflammation. However, in chronic neurodegeneration when disease becomes more widespread, involving distant regions of the brain and periphery, a growing body of evidence suggests that the brain-resident microglia are not only activated (9 -11) but happen to be "primed" by the systemic inflammation, leading to the exaggerated synthesis of proinflammatory molecules (9,(12)(13)(14)(15). Earlier we have shown that microglial activation plays a critical role in the development of PD in mice and monkeys (10, 16 -18). In numerous studies, we and others have shown that microglial cells can be activated by the chronic infiltration of peripheral inflammatory T cells (19 -21). Accordingly, Brochard et al. (5) have shown that both CD8 ϩ and CD4 ϩ T cells significantly invade the SNpc in post-mortem specimens from patients with PD and in MPTP-intoxicated mice. They (5) have also demonstrated that removal of CD4 ϩ , but not of CD8 ϩ , T cells in mice greatly reduced MPTP-induced nigrostriatal dopamine cell death. According to Gendelman and co-workers (6), although Th17 cells exacerbate nigrostriatal dopaminergic neurodegeneration, regulatory T cells attenuate such neurodegeneration.
Although mechanisms leading to the infiltration of T cells into the CNS are poorly understood, recently we have seen marked up-regulation of RANTES and eotaxin, chemokines that are involved in the infiltration of T cells and other immune cells, in vivo in the SNpc and in the serum of MPTP-intoxicated monkey (7), suggesting that these chemokines may participate in nigrostriatal degeneration. Accordingly, here we demonstrate rapid up-regulation of RANTES and eotaxin in nigra and serum of MPTP-intoxicated mice. Furthermore, we also delineate that RANTES and eotaxin are up-regulated in the SNpc of post-mortem PD brains as compared with age-matched controls and that functional blocking antibodies against RANTES and eotaxin protect against nigrostriatal degeneration in MPTP-intoxicated mice. These results suggest that neutralization of RANTES and eotaxin may be beneficial for PD patients.

Rapid Induction of RANTES and Eotaxin in Nigra and Serum of MPTP-intoxicated
Mice-To investigate the role of RANTES and eotaxin in the loss of invaluable dopaminergic neurons in MPTP-intoxicated mice, first we examined whether the expression of these chemokines was induced in midbrains of affected mice. It is evident from Fig. 1A that MPTP intoxication led to time-dependent induction of RANTES and eotaxin mRNA expression in the SNpc. This induction was evident as early as 4 h of MPTP insult (Fig. 1A). However, the expression of RANTES and eotaxin decreased at 72 h of MPTP intoxication (Fig.  1A). These results were confirmed by real time PCR (Fig. 1, B and C). Similarly, Western blotting results showed the induction of RANTES and eotaxin proteins in the nigra (Fig. 1, D-F). Although RANTES was visible from 12 h of MPTP insult, a significant increase in eotaxin was observed at 8 h (Fig. 1, E and  F). These results were also corroborated by ELISA of RANTES and eotaxin in nigral homogenates (Fig. 1, G and H). Next, we monitored the levels of these chemokines in serum. Although an increase in RANTES was visible in serum from 12 h of MPTP intoxication and maximal at 24 h, the eotaxin increase was prominent from 8 h and maximal at 12 h (Fig. 1, I and J).
Microglia in the Nigra of MPTP-intoxicated Mice and Postmortem PD Brains Express RANTES and Eotaxin-Because MPTP intoxication induced the level of RANTES and eotaxin in the nigra, next we wished to identify the cell type that produced these chemokines in the nigra. Recently, chronic microglial activation is becoming a hallmark of different neurodegenerative disorders including PD (17,18,(22)(23)(24). Therefore, we examined whether microglia were capable of producing these FIGURE 1. Rapid up-regulation of RANTES and eotaxin in nigra and serum of MPTP-intoxicated mice. Male C57/BL6 mice (6 -8 weeks old) were insulted with 20 mg/kg of body weight MPTP (four injections at every 2-h interval). After 4,8,12,24, and 72 h of MPTP intoxication, the mRNA expression of RANTES and eotaxin in nigra was monitored by semiquantitative RT-PCR (A) and real time PCR (B, RANTES; C, eotaxin). The protein expression of RANTES and eotaxin was monitored by Western blotting (D). Actin was run as a control. Bands were scanned and values (E, RANTES/actin; F, eotaxin/actin) are presented as relative to control (Con). Levels of RANTES and eotaxin were also measured in nigral homogenates by ELISA (G, RANTES; H, eotaxin). Levels of RANTES and eotaxin were also measured in serum by ELISA (I, RANTES; J, eotaxin). Results are mean Ϯ S.E. of four mice (n ϭ 4) per group. a, p Ͻ 0.001 versus control; b, p Ͻ 0.05 versus control. Error bars represent S.E. chemokines in the nigra of MPTP-intoxicated mice. 24 h after the last injection of MPTP, nigral sections were double labeled for Iba1 and RANTES. As evident from Fig. 2, A-C, MPTP intoxication led to marked induction of RANTES in the nigra, and most of these RANTES signals colocalized with Iba1 ( Fig.  2A). In addition, some RANTES signals also colocalized with
Next, to understand the role of RANTES and eotaxin in nigrostriatal degeneration in PD, nigral sections from post- mortem PD brains and age-matched individuals were immunolabeled for RANTES and eotaxin. Because microglia are the major cell type in the nigra of MPTP-intoxicated mice that express these chemokines, sections were double labeled for RANTES/eotaxin and Iba1. Levels of both RANTES and eotaxin were markedly higher in the nigra of PD brain compared with age-matched controls (Fig. 4, A-D). We also noticed greater Iba1 expression (microglial activation) in the nigra of PD compared with age-matched controls (Fig. 4, A and C). Iba1-positive cells were also positive for both RANTES (Fig. 4A) and eotaxin (Fig. 4C) in the nigra of PD subjects.
Functional Blocking Antibodies against RANTES and Eotaxin Suppress the Infiltration of T Cells into the Nigra and Attenuate the Expression of Proinflammatory Molecules in the Nigra of MPTP-intoxicated Mice-Because we observed a rapid increase in RANTES and eotaxin in the serum of MPTP-intoxicated mice, to understand the role of these chemokines in nigrostriatal degeneration, mice were treated once with a combination of functional blocking antibodies against both RANTES and eotaxin via intraperitoneal (i.p.) injection (Fig.  5A). Chemokines like RANTES and eotaxin are known to induce the migration and homing of inflammatory lymphoid cells such as T cells and monocytes in the site of inflammation. Because substantia nigra is a primary target of neurodegeneration in PD, we determined whether MPTP intoxication induced the infiltration of inflammatory T cells in the nigra. Our dual immunofluorescence analyses of CD4 (green) and tyrosine hydroxylase (TH) (red) clearly displayed a typical CD4-immunoreactive inflammatory cuffing in the nigra of MPTP-intoxicated, but not control, mice (Fig. 5, B and C). This is consistent with that observed in the nigra of PD patients (5). Recently, we have demonstrated infiltration of CD8 ϩ T cells into the nigra of hemiparkinsonian monkeys (7). Therefore, we also analyzed infiltration of CD8 ϩ T cells and found CD8 ϩ inflammatory cuffing in the nigra of MPTP-insulted mice (Fig. 6, A and B). However, as compared with CD4 ϩ T cells, nigral infiltration of CD8 ϩ cells was much lesser in MPTP-intoxicated mice (Figs. 5, B and C, and 6, A and B). Nevertheless, treatment with neutralizing antibodies against RANTES and eotaxin strongly suppressed the infiltration of both CD4 ϩ (Fig. 5, B and C) and CD8 ϩ (Fig. 6, A and B) T cells in the nigra of MPTP-intoxicated mice. These results were specific as normal IgG had no such inhibitory effect (Figs. 5 and 6). These results suggest that the infiltration of peripheral lymphocytes into the nigra of MPTPinsulted mice depends on RANTES and eotaxin.
Infiltration of inflammatory T cells into the site of injury eventually triggers the production of a wide range of proinflammatory molecules (5,8,19,22). Because neutralizing antibodies against RANTES and eotaxin inhibited the infiltration of T cells in vivo in the nigra of MPTP-intoxicated mice, we examined whether these antibodies were able to suppress the expression of various proinflammatory molecules in the nigra. As shown by semiquantitative RT-PCR (Fig. 7A) and quantitative real time PCR (Fig. 7, B-E) experiments, MPTP intoxication led to a marked increase in mRNA expression of iNOS, IL-1␤, GFAP (astroglial marker), and CD11b (microglial marker) in the mid- brain. However, neutralizing antibodies against RANTES and eotaxin, but not control IgG, strongly inhibited MPTP-induced expression of iNOS (Fig. 7, A and B), IL-1␤ (Fig. 7, A and C), GFAP (Fig. 7, A and D), and CD11b (Fig. 7, A and E) mRNAs in vivo in the nigra. Similarly, Western blotting results also show increases in iNOS, IL-1␤, GFAP, and Iba1 in the nigra by MPTP insult and attenuation of these proinflammatory markers (Fig.  7, F-J) by treatment with neutralizing antibodies against RANTES and eotaxin. Double label immunofluorescence analysis also shows that MPTP intoxication led to a marked increase in nigral iNOS protein expression and that this iNOS colocalized strongly with Iba1-positive microglia (Fig.  8A) and partly with GFAP-positive astroglia (Fig. 8B). Similar to mRNA and Western blotting results, treatment of MPTP-intoxicated mice with neutralizing antibodies against RANTES and eotaxin, but not control IgG, led to the suppression of iNOS protein (Fig. 8, A-C). Recently, glial activation is being considered as a pathological hallmark in PD and other neurodegenerative disorders (17,18,22,23). As evident from immunofluorescence analysis of Iba1 and GFAP in nigral sections, MPTP intoxication led to an increase in nigral Iba1 and GFAP protein expression, and neutralizing antibodies against RANTES and eotaxin suppressed MPTP-induced expression of Iba1 (Fig. 8, A and D) and GFAP (Fig. 8, A and E). These results suggest that neutralization of RANTES and eotaxin suppresses the expression of proinflammatory molecules and reduces glial activation in the nigra of MPTP-intoxicated mice.

Functional Blocking Antibodies against RANTES and Eotaxin Protect against MPTP-induced Neurodegeneration-
Because neutralization of RANTES and eotaxin inhibited glial activation and associated neuroinflammation in the nigra of MPTP-intoxicated mice, next we investigated whether functional blocking antibodies against RANTES and eotaxin protected the nigrostriatum from MPTP insult. Mice were treated once with functional blocking antibodies against RANTES and eotaxin 2 h after the last injection of MPTP, and 7 days after the last injection of MPTP, the status of nigral TH neurons and striatal TH fibers was monitored. MPTP intoxication led to an ϳ65% loss of SNpc TH-positive neurons (Fig. 9, A and B) compared with saline-injected controls. However, in MPTP-injected mice treated with functional blocking antibodies against RANTES and eotaxin, the reduction in SNpc TH-positive neurons was about 22% (Fig. 9, A and B). In contrast, no such protective effects were seen in MPTP-intoxicated mice that were treated with control IgG (Fig. 9, A and B). Results were also corroborated by TH Western blotting data of nigral homogenates (Fig. 9, C and D). Similar to the loss of nigral TH neurons, MPTP intoxication led to an ϳ68% reduction of striatal TH optical densities (Fig. 10, A and B) compared with saline-injected controls. Again, marked protection of striatal TH fibers was noted in MPTP-injected mice treated with neutralizing antibodies against RANTES and eotaxin (Fig. 10, A and B). To determine whether neutralization of RANTES and eotaxin protects against biochemical deficits caused by MPTP, we quantified the levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striata 7 days after the MPTP treatment. MPTP intoxication led to a marked decrease in striatal dopamine (Fig. 10C), DOPAC (Fig. 10D), and HVA (Fig. 10E) compared with striata of saline-injected mice. In contrast, MPTP-intoxicated animals that received one injection of neutralizing antibodies against RANTES and eotaxin showed only a 10 -20% loss in striatal dopamine, DOPAC, and HVA (Fig. 10, C-E). Conversely, such protection was not seen in the case of control IgG treatment (Fig. 10, C-E).
Functional Blocking Antibodies against RANTES and Eotaxin Improve Locomotor Functions in MPTP-intoxicated Mice-The ultimate therapeutic goal of neuroprotection in PD is to decrease functional impairment. Therefore, to examine whether neutralization of RANTES and eotaxin protects not only against structural and neurotransmitter damage but also against functional impairments caused by MPTP, we monitored bradykinesia by pole test and locomotor functions by rotarod and open field activities. MPTP insult caused a marked decrease in rotarod performance (Fig. 11A), pole test (Fig. 11B), number of movements (Fig. 11C), movement time (Fig. 11D), horizontal activity (Fig. 11F), total distance (Fig. 11G), and stereotypy (Fig. 11H). In contrast, MPTP insult increased the rest time (Fig. 11E). However, neutralizing antibodies against RANTES and eotaxin significantly improved MPTPinduced hypolocomotion and bradykinesia (Fig. 11, A-H).

Discussion
PD is a progressive age-related neurodegenerative disease with unclear etiology. This disease sometimes progresses ruthlessly, leaving its victims bound to the wheelchair or dependent on caregivers. Despite intense investigations, effective therapy against PD is still unavailable. Administration of a dopamine agonist or levodopa has been the standard treatment for PD. However, it is often associated with a number of side effects and unsatisfactory outcomes. Therefore, understanding the mechanism of the disease process of PD and development of an effective neuroprotective therapeutic approach to halt the disease progression are of paramount importance. Here, we have demonstrated a rapid increase in RANTES and eotaxin in the nigra and serum of mice upon MPTP intoxication. Although microglia were the main producers of RANTES and eotaxin in the SNpc of MPTP-intoxicated mice, we also noticed some RANTES and eotaxin in GFAP-positive astrocytes. Furthermore, an increase in RANTES and eotaxin in the SNpc of postmortem PD brains as compared with age-matched controls suggests that these chemokines may play a role in the loss of nigral dopaminergic neurons in PD.
Because the increase in RANTES and eotaxin was also seen in the serum of MPTP-intoxicated mice, we used functional blocking antibodies to neutralize their activities in the periphery. Several lines of evidence clearly suggest that peripheral administration of functional blocking antibodies against RANTES and eotaxin reduces inflammation and protects the nigrostriatum in MPTP-intoxicated mice. Our conclu- sion is based on the following findings. First, inflammation plays a role in the pathogenesis of nigrostriatal degeneration in PD patients and MPTP-intoxicated mice (12,17,18,23,24). Accordingly, MPTP insult increased the expression of different proinflammatory cytokines (TNF␣, IL-1␤, and IL-6) and iNOS in the SNpc. However, i.p. injection of a combination of antibodies against RANTES and eotaxin markedly decreased the expression of these proinflammatory molecules in the SNpc of MPTP-intoxicated mice. Second, as expected, MPTP intoxication led to glial activation in the nigra as evident by a marked increase in expression of GFAP and CD11b, which was inhibited by treatment with antibodies against RANTES and eotaxin. Third, as observed in PD, nigral dopaminergic neurons disappeared in MPTP-intoxicated mice. However, treatment with antibodies against RANTES and eotaxin protected TH-positive dopaminergic neurons from MPTP toxicity. Fourth, treatment with antibodies against RANTES and eotaxin also protected striatal TH fibers from MPTP toxicity and restored the level of neurotransmitters. Lastly, antibody treatment ameliorated functional impairment in MPTP-intoxicated mice. We did not notice any side effects (e.g. hair loss, weight loss, untoward infection, etc.) in any of the mice used during the course of the study, suggesting that antibodies against RANTES and eotaxin may not exhibit any side effects.
RANTES (25) and eotaxin (26) are two important proinflammatory chemokines that are produced by T cells and antigenpresenting cells such as macrophages and microglia. RANTES, a 68-amino acid-long polypeptide, is known to induce the migration and homing of classical lymphoid cells such as T cells and monocytes and other immune cells including basophils, eosinophils, natural killer cells, dendritic cells, and mast cells (27). Similarly, eotaxin, another small 71-amino acid-long chemokine, is capable of inducing infiltration of mononuclear cells in the site of inflammation (28). Therefore, a major function of these two chemokines is to control the homing of T cells. A few earlier studies have already reported infiltration of T cells into the nigra of an MPTP mouse model (5) and MPTP-intoxicated rhesus monkeys (7). Here, we also saw marked infiltration of CD4 ϩ and relatively less infiltration of CD8 ϩ T cells into the nigra upon MPTP intoxication. Interestingly, neutralization of RANTES and eotaxin strongly inhibited the infiltration of both CD4 ϩ and CD8 ϩ T cells into the nigra of MPTP-intoxicated mice, suggesting that MPTP insult induces infiltration of T cells into the nigra via RANTES and eotaxin.
Although whether T cell infiltration is primary or secondary to nigrostriatal degeneration is still unclear, once T cells infiltrate into the nigra, there are several direct and indirect pathways by which T cells could influence dopaminergic neurodegeneration. For example, it has been reported that the migration of antigen-specific CD4 ϩ T cells from the periphery to the CNS generates immunocyte-microglia activities that perpetuate neuroinflammation and affect neuronal survival (29). Earlier we have shown that effector T cells are capable of activating microglia for the production of various proinflammatory molecules via cell-to-cell contact (19,30). This contact process involves VLA4 (␣4␤1) integrin of T cells and VCAM1 of microglia (19,31). Furthermore, activated T cells may also activate microglia via CD40-CD40 ligation (32)(33)(34). According to Nitsch et al. (35), the cytotoxic T cell-mediated lethal increase in neuronal calcium could be prevented by blocking both perforin and glutamate receptors.
In summary, we have demonstrated that MPTP intoxication leads to a rapid increase in RANTES and eotaxin in the SNpc and serum of mice and that neutralization of these two chemokines protects nigral dopaminergic neurons. Although the MPTP mouse model does not recapitulate all the features of PD in humans, RANTES and eotaxin are also up-regulated in the nigra of post-mortem PD brains as compared with control brains. Therefore, our results suggest that neutralizing antibodies against RANTES and eotaxin may have therapeutic efficacy in PD.

Materials and Methods
Reagents-Mouse RANTES and eotaxin ELISA kits were purchased from R&D Systems (Minneapolis, MN). Anti-CD3, -CD4, and -CD8 antibodies were purchased from eBioscience. Rabbit anti-TH antibody was purchased from Millipore. Anti-Iba1 antibody was purchased from Abcam. Cy2-and Cy5-conjugated antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Animals and MPTP Intoxication-6 -8-week old C57BL/6 mice were purchased from Harlan (Indianapolis, IN). Animal maintenance and experiments were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use committee of the Rush University Medical Center (Chicago, IL). For acute MPTP intoxication, mice received four i.p. injections of MPTP-HCl (18 mg/kg free base; Sigma) in saline at 2-h intervals (12,17,18,36,37). Control animals received only saline.
Human Brain Tissue-Autopsy brain tissues from four male PD patients and four control subjects were obtained from the Rush PD Center Brain Bank. PD patients and control subjects did not differ significantly for their mean age at death (PD, 74 Ϯ 3 years; control, 79 Ϯ 18 years). The mean post-mortem intervals for PD and controls were 4.1 Ϯ 0.8 and 10.9 Ϯ 1.1 h, respectively.
Treatment of MPTP-intoxicated Mice with Neutralizing Antibodies against RANTES and Eotaxin-Azide-free neutralizing antibodies against RANTES/CCL5 and eotaxin/CCL11 were obtained from R&D Systems. 2 h after the last injection of MPTP, mice were treated once with the combination of anti-RANTES Ab (20 g/mouse) and anti-eotaxin Ab (20

Targeting RANTES and Eotaxin in MPTP Mouse Model
g/mouse) via i.p. injection. Antibodies were reconstituted in sterile PBS in such a way that total injection volume remained at 100 l/mouse. A group of MPTP-intoxicated mice also received IgG (40 g/mouse) as a control via i.p. injection.
Semiquantitative RT-PCR Analysis-Total RNA was isolated from nigra using Ultraspec-II RNA reagent (Biotecx Laboratories, Inc., Houston, TX) following the manufacturer's protocol. To remove any contaminating genomic DNA, total RNA was digested with DNase. RT-PCR was carried out as described earlier (17,38,39)  Real Time PCR Analysis-DNase-digested RNA was analyzed by real time PCR in the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) as described earlier (17, 38 -40) using TaqMan Universal Master Mix and optimized concentrations of FAM-labeled probes and primers. Data were processed using ABI Sequence Detection System 1.6 software.
Western Blotting Analysis-Immunoblotting analysis for RANTES, eotaxin, iNOS, IL-1␤, GFAP, Iba1, and TH was carried out as described earlier (41)(42)(43). Briefly, cell homogenates were electrophoresed, proteins were transferred onto a nitrocellulose membrane, and bands were visualized with an Odyssey infrared scanner after immunolabeling with respective primary antibodies followed by an infrared fluorophore-tagged secondary antibody (Invitrogen).
Immunohistochemistry and Quantitative Morphology-Seven days after MPTP intoxication, mice were sacrificed, and their brains were fixed, embedded, sectioned (30 m thick), and processed for TH and thionin staining as described previously (18,22). Total numbers of TH-and Nissl-stained neurons in SNpc were counted stereologically with STEREO INVESTIGA-TOR software (MicroBrightfield, Williston, VT) by using an optical fractionator (18,22). Quantitation of striatal TH immunostaining was performed as described (18,22). Optical density measurements were obtained by digital image analysis (Scion, Frederick, MD). Striatal TH optical density reflected dopaminergic fiber innervation.
For immunofluorescence staining on fresh-frozen nigral sections isolated from mice after 1 day of MPTP insult, goat antimouse RANTES (1:100), rat anti-mouse eotaxin (1:100), rabbit anti-mouse Iba1 (1:100), goat anti-mouse GFAP (1:100), mouse anti-mouse CD4 (1:100), and mouse anti-mouse iNOS (1:250) were used. The samples were mounted and observed under an Olympus IX81 fluorescence microscope. Counting analysis was performed using Olympus MicroSuite V software with the help of a touch counting module. After acquiring images under 20ϫ objective lens, images were further analyzed as follows. Before counting cells, the entire image area was calibrated with the help of a rectangular box available in the touch counting panel.
Once the area of the image was measured, the touch counting program was applied to count the number of fluorescence signals using a simple mouse click method. Next, the total number of signals in a given area was divided by the total area of the image and presented as number of cells per square millimeter unit.
HPLC Analysis for Measurement of Striatal Dopamine and Its Metabolite Levels-Striatal levels of dopamine, DOPAC, and HVA were quantified as described earlier (10,12,18,22,36). Briefly, mice were sacrificed by cervical dislocation after 7 days of MPTP intoxication, and their striata were collected, immediately frozen in dry ice, and stored at Ϫ80°C until analysis. On the day of the analysis, tissues were sonicated in 0.2 M perchloric acid containing isoproterenol, and resulting homogenates were centrifuged at 20,000 ϫ g for 15 min at 4°C. After pH adjustment and filtration, 10 l of supernatant was injected onto a Eicompak SC-3ODS column (complete stand-alone HPLCelectrochemical detection system EiCOMHTEC-500, JM Science Inc., Grand Island, NY) and analyzed following the manufacturer's protocol.
Behavioral Analyses-Three types of behavioral experiments were conducted. These included an open field experiment for locomotor activity, a pole test for bradykinesia, and a rotarod experiment for feet movement as described earlier (12,17,18,36,44). Locomotor activity was measured 7 days after the last dose of injected MPTP in a Digiscan monitor (Omnitech Electronics, Inc., Columbus, OH). This Digiscan monitor records stereotypy and rearing, behaviors that are directly controlled by the striatum, as well as other basic locomotion parameters such as horizontal activity, total distance traveled, number of movements, movement time, rest time, mean distance, mean time, and center time. Before any insult or treatment, mice were placed inside the Digiscan infrared activity monitor for 10 min daily and on the rotarod for 10 min daily for 3 consecutive days to train them and record their baseline values. Briefly, animals were removed directly from their cages and gently placed nose first into a specified corner of the open field apparatus, and after release, data acquisition began at every 5-min interval. Digiscan software was used to analyze and store horizontal and vertical activity data, which were monitored automatically by infrared beams. Bradykinesia was measured by the time to turn headdown and completely descend a wooden pole wrapped in cloth tape. Briefly, mice were acclimatized to the pole (1-cm diameter, 40-cm height) over three trials of 120 s each. Each trial was separated by 60 s, and during behavioral testing, each mouse was tested thrice. In the rotarod, the feet movement of the mice was observed at different speeds. To eliminate stress and fatigue, mice were given a 5-min rest interval. JULY 15, 2016 • VOLUME 291 • NUMBER 29

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Statistical Analysis-All values are expressed as means Ϯ S.E. Differences among means were analyzed by one-way analysis of variance or Kruskal-Wallis test (comparison among all four groups) and post hoc pairwise comparison. In other cases, twosample t tests were also used to compare control versus MPTP and MPTP versus antibody.
Author Contributions-G. C. contributed to design of experiments, acquisition of data, and analysis and interpretation of data. S. B. R. contributed to acquisition and analysis of data. A. R. contributed to acquisition and analysis of data. J. H. K. contributed to data acquisition, analysis of data, and manuscript writing. K. P. conceived and designed experiments and analyzed and interpreted data.