β2-Adrenergic Receptor Activation Prevents Rodent Dopaminergic Neurotoxicity by Inhibiting Microglia via a Novel Signaling Pathway This information is current as of September 29, 2021. Li Qian, Hung-ming Wu, Shih-Heng Chen, Dan Zhang, Syed F. Ali, Lynda Peterson, Belinda Wilson, Ru-Band Lu, Jau-Shyong Hong and Patrick M. Flood J Immunol published online 18 February 2011 http://www.jimmunol.org/content/early/2011/02/16/jimmun Downloaded from ol.1002449

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 18, 2011, doi:10.4049/jimmunol.1002449 The Journal of Immunology

b2-Adrenergic Receptor Activation Prevents Rodent Dopaminergic Neurotoxicity by Inhibiting Microglia via a Novel Signaling Pathway

Li Qian,*,† Hung-ming Wu,† Shih-Heng Chen,† Dan Zhang,† Syed F. Ali,‡ Lynda Peterson,* Belinda Wilson,† Ru-Band Lu,x Jau-Shyong Hong,† and Patrick M. Flood*

The role of the b2 adrenergic receptor (b2AR) in the regulation of chronic neurodegenerative inflammation within the CNS is poorly understood. The purpose of this study was to determine neuroprotective effects of long-acting b2AR agonists such as salmeterol in rodent models of Parkinson’s disease. Results showed salmeterol exerted potent neuroprotection against both LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium–induced dopaminergic neurotoxicity both in pri- Downloaded from mary neuron-glia cultures (at subnanomolar concentrations) and in mice (1–10 mg/kg/day doses). Further studies demonstrated that salmeterol-mediated neuroprotection is not a direct effect on neurons; instead, it is mediated through the inhibition of LPS- induced microglial activation. Salmeterol significantly inhibited LPS-induced production of microglial proinflammatory neuro- toxic mediators, such as TNF-a, superoxide, and NO, as well as the inhibition of TAK1-mediated phosphorylation of MAPK and p65 NF-kB. The anti-inflammatory effects of salmeterol required b2AR expression in microglia but were not mediated through the conventional G protein-coupled receptor/cAMP pathway. Rather, salmeterol failed to induce microglial cAMP production, http://www.jimmunol.org/ could not be reversed by either protein kinase A inhibitors or an exchange protein directly activated by cAMP agonist, and was dependent on b-arrestin2 expression. Taken together, our results demonstrate that administration of extremely low doses of salmeterol exhibit potent neuroprotective effects by inhibiting microglial cell activation through a b2AR/b-arrestin2–dependent but cAMP/protein kinase A-independent pathway. The Journal of Immunology, 2011, 186: 000–000.

he b2 adrenergic receptor (b2AR) is a G protein-coupled elucidated by the development of short-acting and long-acting receptor (GPCR) that is known to regulate a variety of b2AR agonists for pharmacological studies and clinical usage. T biological functions, including the regulation of smooth Numerous studies using these b2AR agonists have shown that by guest on September 29, 2021 muscle activity in the airway and vasculature. b2AR expression they possess the ability to regulate inflammatory responses by has been identified on immune cells such as macrophages, immune cells, and several of these agonists such as salmeterol microglia, T cells, and B cells, and signaling through this receptor (Advair) and formoterol (Symbicort) are already being used as can influence the inflammatory response of these cells (1–7). The anti-inflammatory therapeutics to treat asthma and chronic ob- functional role of b2AR in biological responses has been further structive pulmonary disease (COPD) (8–10). However, potential use of b2AR agonists in neurodegenerative diseases in the CNS *North Carolina Oral Health Institute, University of North Carolina at Chapel Hill, has not been well studied. Chapel Hill, NC 27599; †Neuropharmacology Section, Laboratory of Toxicology and It is now well accepted that inflammation plays a major role in Pharmacology, National Institute of Environmental Health Sciences, National Insti- the progression of a number of neurodegenerative disorders, in- tutes of Health, Research Triangle Park, NC 27709; ‡Neurochemistry Laboratory, National Center for Toxicological Research/Food and Drug Administration, Jeffer- cluding Parkinson’s disease (PD), Alzheimer’s disease, multiple son, AR 72079; and xDepartment of Psychiatry, College of Medicine and Hospital, sclerosis, and AIDS-related dementia (11). PD is characterized by National Cheng Kung University, Tainan 70428, Taiwan the progressive loss of dopaminergic (DA) neurons within the Received for publication July 22, 2010. Accepted for publication January 24, 2011. substantia nigra (SN), which results in movement disorders as well This work was supported by National Institutes of Health Grant DE-13079 from the as psychological/psychiatric changes in diseased individuals. Al- National Institute for Dental and Craniofacial Research and a grant from the Michael J. Fox Foundation. This work was also supported in part by the Intramural Research though the disease mechanisms that ultimately cause PD are still Program of the National Institutes of Health/National Institute on Environmental unclear, it is believed that the progressive nature of PD is char- Health Sciences. acterized by chronic inflammation-induced DA neurodegeneration Address correspondence and reprint requests to Dr. Patrick M. Flood, North Carolina within the SN (12–14). The premise of microglial involvement Oral Health Institute, University of North Carolina at Chapel Hill, CB #7454, Chapel Hill, NC 27599-7454. E-mail address: pat_fl[email protected] and the elevation of proinflammatory mediators in PD have been supported by the analysis of post mortem brains from PD patients The online version of this article contains supplemental material. that provide clear evidence of microglial activation in the SN (15– Abbreviations used in this article: b2AR, b2 adrenergic receptor; COPD, chronic obstructive pulmonary disease; DA, dopaminergic; DCFH-DA, dichlorodihydrofluor- 17). Using various PD animal models, we and others have shown escein diacetate; DOPAC, 3,4-dihydroxyphenylacetic acid; EPAC, exchange protein that microglia, the major immune cell of the CNS containing high directly activated by cAMP; GPCR, G protein-coupled receptor; HVA, homovanillic levels of b2AR (18), play a critical role in mediating toxicity of acid; iROS, intracellular reactive oxygen species; MPP+, 1-methyl-4-phenylpyridi- nium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s dis- DA neurons in the SN area. Recent studies have shown that ac- ease; PKA, protein kinase A; ROS, reactive oxygen species; siRNA, small tivation of nigral microglia and the subsequent release of neuro- interfering RNA; SN, substantia nigra; SNpc, SN pars compacta; SOD, superoxide a dismutase; TH, tyrosine hydroxylase; TH-IR, tyrosine hydroxylase-immunoreactive; toxic factors, including the proinflammatory cytokine TNF- ,as WST-1, water-soluble tetrazolium salt-1. well as NO and reactive oxygen species (ROS), are considered key

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002449 2 SALMETEROL-MEDIATED NEUROPROTECTION mediators of DA neurodegeneration in PD (11, 19, 20). Because microglia, 48% astrocytes, 41% neurons, and 1% TH-immunoreactive most long-acting b2AR agonists are highly lipophilic and should (TH-IR) neurons. The composition of the neuron-glia cultures of mice is readily gain access to the brain, it is likely that these compounds very similar to that of rats, which consists of 12% microglia, 48% astro- cytes, 40% neurons, and 1% TH-IR neurons. could have an immunomodulatory effect on the progression of inflammation in PD patients by inhibiting the activation of Primary mesencephalic neuron-enriched cultures microglia. Therefore, we hypothesize that b2AR agonists might Midbrain neuron-enriched cultures were established as described previously well serve as a new class of compounds that could be used as an (23). Briefly, 24 h after seeding, cytosine b-D-arabinocide was added to anti-inflammatory treatment for PD. a final concentration of 10 mM to suppress glial proliferation. Three days In this study, we report that long-acting b2AR agonists have later, the media were removed and replaced with maintenance medium. anti-inflammatory and DA-neuroprotective properties, even at Cells were used for drug treatments 7 d after initial seeding. Routinely, the 7-d-old neuron-enriched cultures, which normally contain ,0.1% micro- extremely low doses, and that administration of the long-acting glia and ,3–5% astrocytes, were used for treatment. Among the neuronal b2AR agonist salmeterol significantly protects DA neurons against population (Neu-N immunoreactive neurons), 2.7–3.9% were DA neurons LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- (TH-IR–positive neurons). induced toxicity both in vitro and in vivo. Mechanistic studies us- Primary midbrain neuron-astroglia cocultures ing primary mesencephalic neuron-glia cultures demonstrated that salmeterol, as well as several other long-acting b2AR agonists, Rat primary neuron-astroglia cocultures were obtained by suppressing exert potent neuroprotection through their inhibition of microglial microglial proliferation with 1.5 mM leucine methyl ester 24 h after seeding inflammatory mediator production. Furthermore, we showed that the cells, as described previously (23). Three days later, cultures were changed back to maintenance medium and used for treatment 7 d after the anti-inflammatory effects of salmeterol seen at these extremely initial seeding. The cultures stained with Iba1 or F4/80 Ab showed ,0.1% Downloaded from low doses requires the presence of b2AR, are mediated through the microglia. inhibition of both MAPK and NF-kB signaling pathways in acti- vated microglia, and function independently of the canonical Primary microglia-enriched cultures GPCR/cAMP/protein kinase A (PKA) signaling pathway. These Mice microglia-enriched cultures with a purity of .98% were prepared results may suggest a new mode of action for b2AR agonists as a from whole brains of 1-d-old mice pups as described previously (24). For superoxide assays, 105 cells were grown overnight in 96-well culture plates therapeutic in the treatment of chronic inflammatory conditions http://www.jimmunol.org/ before use. of the CNS. 3H-DA uptake assay Materials and Methods 3H-DA uptake assays were performed as described previously (24). Briefly, Animals cells were incubated for 20 min at 37˚C with 1 mM 3H-DA in Krebs- Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, 1.8 mM CaCl , 1.2 mM MgSO , 1.3 mM EDTA, and 5.6 mM glucose [pH ME). Timed-pregnant Fisher F344 rats and C57/BL6 mice were obtained 2 4 7.4]). Cells were washed with ice-cold Krebs-Ringer buffer three times, from Charles River Laboratories (Raleigh, NC). Congenic BALB/c b2AR- after which, the cells were collected in 1 N NaOH. Radioactivity was deficient mice were derived from FVB b AR-deficient mice (Taconic, 2 determined by liquid scintillation counting. Nonspecific DA uptake ob- Hudson, NY). Housing and breeding of the animals were performed in by guest on September 29, 2021 served in the presence of mazindol (10 mM) was subtracted. strict accordance with the National Institutes of Health guidelines. Reagents Immunocytochemistry Salmeterol was obtained from Tocris Cookson (Ballwin, MO). Rp-cAMPS Immunostaining was performed as described previously (22). Briefly, and cAMP Enzyme Immunoassay Kit was purchased from BIOMOL formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogen per- (Plymouth Meeting, PA). Long-acting compounds including formoterol oxide, followed by sequential incubation with blocking solution, after fumarate dehydrate, bambuterol hydrochloride, and clenbuterol hydro- which, the cells were incubated overnight at 4˚C with Abs against TH (1/ chloride, as well as MPTP and 1-methyl-4-phenylpyridinium (MPP+), 20,000). Cells were incubated with biotinylated secondary Ab for 2 h, were purchased from Sigma-Aldrich (St. Louis, MO). LPS used for in vitro followed by incubation with avidin/biotin complex reagents for 40 min. 9 studies (Escherichia coli strain O111:B4) was purchased from Calbiochem Color was developed with 3,3 -diaminobenzidine. For morphological (San Diego, CA), and LPS used for in vivo studies was purchased from analysis, the images were recorded with an inverted microscope (Nikon, Sigma-Aldrich. Cell culture reagents were obtained from Invitrogen Tokyo, Japan) connected to a charge-coupled device camera (DAGE-MTI, (Carlsbad, CA). 3H-DA (30 Ci/mmol) was obtained from PerkinElmer Life Michigan City, IN) operated with the MetaMorph software (Universal Sciences (Boston, MA). The polyclonal anti-tyrosine hydroxylase (TH) Ab Imaging, Downingtown, PA). For visual counting of TH-IR neurons, nine was a gift from Dr. J. Reinhard (GlaxoSmithKline, Research Triangle Park, representative areas per well of the 24-well plate were counted under the 3 NC). The Vectastain avidin/biotin complex kit and biotinylated secondary microscope at 100 magnification by three individuals. The average of Abs were purchased from Vector Laboratories (Burlingame, CA). The these scores was reported. fluorescence probe dichlorodihydro-fluorescein diacetate (DCFH-DA) was obtained from Calbiochem (La Jolla, CA). Anti–phospho-ERK1/2 Ab, Superoxide assay anti-ERK1/2 Ab, anti–phospho-p38 Ab, anti-p38 Ab, anti–phospho-JNK The production of superoxide was determined by measuring the superoxide Ab or anti-JNK Ab, anti–phospho-p65 or anti-p65 Ab, and anti-TAK1 Ab dismutase (SOD)-inhibitable reduction of water-soluble tetrazolium salt-1 were purchased from Cell Signaling Technology (Danvers, MA). Negative b (WST-1) (25, 26). Neuron-glia or microglia-enriched cultures in 96-well control small interfering RNA (siRNA) and -arrestin2 siRNA are from culture plates were washed twice with HBSS without phenol red. Cultures Invitrogen (Carlsbad, CA). were then incubated at 37˚C for 30 min with vehicle control (DMSO) or Primary mesencephalic neuron-glia cultures salmeterol in DMSO (50 ml/well). Then, 50 ml HBSS with and without SOD (final concentration, 50 U/ml) was added to each well along with 50 Neuron-glia cultures were prepared from the ventral mesencephalic tissues ml WST-1 (1 mM) in HBSS and 50 ml vehicle or LPS (10 ng/ml). To of embryonic day 14–15 rats or day 13–14 mice, as described previously measure superoxide production induced by MPP+, 7-d-old mesencephalic (21, 22). Briefly, dissociated cells were seeded at 1 3 105/ and 5 3 105/ neuron-glia cultures grown in 96-well plates were treated with salmeterol + well in poly-D-lysine–coated 96- and 24-well plates, respectively. Cells in the presence and absence of MPP or vehicle alone in 150 ml phenol were maintained at 37˚C in a humidified atmosphere of 5% CO2 and 95% red-free treatment medium. Four days after treatment, 50 ml HBSS with air in MEM containing 10% FBS, 10% horse serum, 1 g/l glucose, 2 mM and without SOD (final concentration, 50 U/ml) was added to each well L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 50 along with 50 ml WST-1 (1 mM) in HBSS. Fifteen minutes later, absor- U/ml penicillin, and 50 mg/ml streptomycin. Seven-day-old cultures were bance at 450 nm was read with a SpectraMax Plus microplate spectro- used for drug treatments. At the time of treatment, immunocytochemical photometer (Molecular Devices, Sunnyvale, CA). The difference in ab- analysis indicated that the rat neuron-glia cultures were made up of 11% sorbance observed in the presence and absence of SOD was considered to The Journal of Immunology 3 be the amount of superoxide produced, and results were expressed as the thanized 21 d after the last MPTP/saline treatment with an i.p. injection of percentage of vehicle-treated control cultures. 120 mg/kg pentobarbital and then perfused through the left ventricle with saline. Mice were divided into two groups: 1) the striatum region was dis- Intracellular ROS assay sected and immediately frozen in dry ice for the analysis of levels of DA and Intracellular ROS (iROS) were determined by using a DCFH-DA assay as its metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] and homo- previously described with minor modifications (27). DCFH-DA enters vanillic acid [HVA]) by HPLC. 2) Saline perfusion was followed by 4% cells passively and is deacetylated by esterase to nonfluorescent DCFH. paraformaldehyde for immunocytochemical analyses. Brains were removed DCFH reacts with ROS to form DCF; the fluorescent product DCFH-DA and postfixed in 4% paraformaldehyde overnight at 4˚C. Brains were then was dissolved in methanol at 10 mM and was diluted 500-fold in HBSS to placed into 30% sucrose/PBS solution at 4˚C until the brains sank to the yield DCFH-DA at 20 mM. The cells were exposed to DCFH-DA for 1 h bottom of the container. Coronal sections including SNpc were cut on m and then treated with HBSS containing the corresponding concentrations a horizontal sliding microtome into 35- m transverse free-floating sections. of LPS for 2 h. The fluorescence was read immediately at wavelengths Analysis of striatal catecholamine content of 485 nm for excitation and 530 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices). The ex- The levels of DA and its metabolites (DOPAC and HVA) were determined perimental value minus the value of the control group was interpreted as by HPLC and coupled with electrochemical detection as described pre- the increase in iROS. viously (30). In brief, striatal tissues were sonicated in 0.2 M perchloric acid (20% w/v) containing the internal standard 3,4-dihydroxybenzylamine Nitrite and TNF-a assays (100 ng/ml). After centrifugation, 150 ml of the supernatant was passed through a 0.2-mm Nylon-66 filter, and 25 ml of the filtrate representing 2.5 The production of NO was determined by measuring the accumulated levels mg striatal tissue was used. The concentrations of DA, DOPAC, and HVA of nitrite in the supernatant with Griess reagent, and the release of TNF-a a2 were calculated using standard curves that were generated by determining, was measured with a mouse TNF- ELISA kit from R&D Systems in triplicate, the ratios between three known amounts of the internal stan- (Minneapolis, MN) as described previously (23). dard. Downloaded from Western blot analysis Rotarod test Enriched microglia were pretreated with vehicle or salmeterol in the The rotorod apparatus (Rotamex, Columbus Instruments, Columbus, OH) presence or absence of LPS, and 15 min later, cells were lysed and collected was used to evaluate the ability of staying on the rotating rod. Mice were m total protein for assay. Equal amounts of protein (20 g/lane) were sep- transported (within their home cage) to acclimate to the testing room for 1 h arated by 4–12% Bis-Tris Nu-PAGE gel and transferred to polyvinylidene prior to testing started. The parameters of the rotor-rod system include start difluoride membranes (Novex, San Diego, CA). For detection of the speed, acceleration, and highest speed (2 rpm, accelerate 2 rpm/10 s, 20 http://www.jimmunol.org/ phosphorylation of MAPK, membranes were blocked with 5% BSA PBS rpm). Each mouse was placed on the confined section of the rod. We then buffer and washed three times, followed by incubation with anti–phospho- turn on the rotarod, which then begins to rotate with a smooth increase in ERK1/2 Ab, anti-ERK1/2 Ab, anti–phospho-p38 Ab, anti-p38 Ab, anti– speed from 2 rpm. Mice received three consecutive trials. The rest period phospho-JNK Ab, anti-JNK Ab, anti–phospho-TAK1 Ab, or anti-TAK1 Ab between each trial was 2 min. The mean latency for the three trials was at 1/1000 dilution (Cell Signaling Technology, Danvers, MA) overnight at used for the analysis. The latency to fall was measured in seconds. In all 4˚C. Anti-rabbit HRP-linked secondary Ab (1/2000 dilution) was incu- trials, if the mouse did not fall from the rod, it was removed from the rod bated for 1 h at 25˚C. The same detection system as above was used. after 2 min. cAMP assay Analysis of neurotoxicity Primary microglia cells were treated with 0 or 1028 M∼10211 M salmeterol

The loss of DA neurons was assessed by counting the number of TH-IR by guest on September 29, 2021 for 5, 10, and 30 min. Cells were lysed in 0.1 M HCl for 10 min, and the neurons following immunostaining of brain sections. Twenty-four con- supernatants were used for cAMP assays following the manufacturer’s secutive brain slices (35 mm thickness), which encompassed the entire protocol of cAMP Enzyme Immunoassay Kit from BIOMOL. The ab- SNpc, were collected. A normal distribution of the number of TH-IR sorbance was measured at 405 nm using a colorimetric 96-well plate neurons in the SNpc was constructed based on the counts of 24 slices reader. Data were expressed as picomoles cAMP per 1 million cells. from C57BL/6J mice. Eight evenly spaced brain slices from saline, LPS- DNA binding assay injected, or salmeterol-treated animals were immunostained with an Ab against TH and counted. The distribution of the cell numbers from each DNA binding assay was performed by using NF-kB (p65) Transcription animal was matched with the normal distribution curve to correct for errors Factor Assay Kit from Cayman Chemical following the instructions. resulting from the cutting. Samples were counted in a double-bind manner with three individuals. Conclusions were drawn only when the difference RNA interference was within 5%. siRNA duplexes with sequences specifically targeting b-arrestin2 RNAwas 59- Microglia counting UGGUGUCCUACAGGGUCAAGGUGAA-39 and negative control siRNA (Invitrogen) were transfected into primary mouse microglia cells using an Microglia were counted as described previously (31). Briefly, three vertical Macrophage Nucleofector (Lonza, Allendale, NJ) and the manufacturer’s and horizontal lines were drawn in equal intervals in each well of the 24-well buffer kit, and protocol was optimized for mouse macrophage. Knockdown plate. For quantification of microglia, nine representative areas (0.9 3 0.7 of expression of the target was determined by Western blotting. mm/field) per well at the line intersections were focused, and 81 fields from three independent experiment in triplicate were selected at 3100 magnifi- Systemic LPS injection model cation by using Nikon Diaphot equipped with Dage-MTI (DC 330) camera with MetaMorph software. The number of Iba-1–positive microglia was Three months after a single systemic LPS injection (5 mg/kg, i.p.) (28, 29), counted. Counting was performed in a double-blind manner by three indi- continuous infusion of extremely low doses (1∼10 mg/kg/day, delivered viduals, and conclusions were drawn only when the differencewas within 5%. s.c. via an Alzet osmotic pump) of salmeterol were administered for 2 wk. Rotarod activity was evaluated at 7 and 10 mo after LPS injection. Ten Statistical analysis months after LPS injection, mice were euthanized, and brains were re- 6 moved and postfixed in 4% paraformaldehyde overnight at 4˚C. Brains The data were presented as the means SE. For multiple comparisons of were then placed into 30% sucrose/PBS solution at 4˚C until the brains groups, two-way or three-way ANOVA was used. Statistical significance sank to the bottom of the container. Coronal sections including SN pars between groups was assessed by paired Student t test, followed by Bon- compacta (SNpc) were cut on a horizontal sliding microtome into 35-mm ferroni correction using the JMP program (SAS Institute, Cary, NC). A p , transverse free-floating sections. value 0.05 was considered statistically significant. MPTP model Results For 6 consecutive days, 8-wk-old C57BL/6 male mice received daily MPTP Low doses of b2AR agonists are neuroprotective against injections (15 mg/kg MPTP·HCl [14.52 mg/kg as a base], s.c.). Salmeterol LPS-induced DA neurotoxicity (1 or 10 mg/kg/day) was given by continuous infusion (via an Alzet mini- pump) for 2 wk starting at 2 d before the first MPTP injection. Mice used Because b2AR agonists have been shown to have potent anti- as controls received an equal volume of 0.9% saline. The mice were eu- inflammatory effects on a number of different chronic inflammatory 4 SALMETEROL-MEDIATED NEUROPROTECTION conditions, we explored whether b2AR agonists are neuropro- encephalic neuron-glia cultures were pretreated with either short- tective against inflammation-induced toxicity of DA neurons (Fig. acting b2AR agonist salbutermol (Fig. 1A) or long-acting b2AR 1). On the basis of the blood t1/2, b2AR agonists were classified agonists bambuterol (Fig. 1B), clenbuterol (Fig. 1C), formoterol into short acting (serum t1/2, 3–4 h) and long acting (serum t1/2, (Fig. 1D), and salmeterol (Fig. 1E–G) at a wide range of con- ∼12 h) (32, 33), and both types were tested in this study. Mes- centrations (1027–10212 M) for 30 min prior to the addition of Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Low doses of b2AR agonists protect DA neurons against LPS-induced toxicity. Rat primary mesencephalic neuron-glia cultures were seeded in a 24-well culture plate at 5 3 105, then pretreated with vehicle or indicated concentrations of b2AR agonists, including short-acting b2AR agonist salbutermol (A), long-acting b2AR agonists bambuterol (B), clenbuterol (C), formoterol (D), and salmeterol (E–G) for 30 min before the addition of 5 ng/ ml LPS. Seven days later, the LPS-induced DA neurotoxicity was quantified by the 3H-DA uptake assay (A–E); the immunocytochemical analysis of salmeterol and LPS-treated cells, including TH-IR neuron counts (F) and the representative pictures of TH+ neuron immunostaining (G). Results in A–E were expressed as a percentage of the vehicle-treated control cultures and were the means 6 SE from three independent experiments in triplicate. *p , 0.05, **p , 0.01 compared with the LPS-treated cultures. Scale bar, 50 mm. The Journal of Immunology 5

FIGURE 2. Salmeterol mediates neuroprotection when administered prior to or after exposure to LPS. Rat primary mesencephalic neuron-glia cultures were seeded in a 24-well culture plate at 5 3 105, and vehicle or salmeterol at 10210 M was added to neuron-glia cultures 30 min prior to, simultaneously with, 30 min, 1 h, 2 h, or 3 h following the addition of LPS. DA neurotoxicity was measured 7 d later. Results were expressed as a Downloaded from percentage of the vehicle-treated control cultures and were the means 6 SE from three independent experiments in triplicate. *p , 0.05, **p , 0.01 compared with the LPS-treated cultures. FIGURE 4. Salmeterol was also capable of inhibiting progressive DA LPS. Seven days later, LPS-induced DA neurotoxicity was quan- neurodegeneration induced by injection of the neurotoxin MPTP. To in- tified by measuring DA neuronal function using the 3H-DA uptake duce DA neurodegeneration, MPTP (15 mg/kg) was injected s.c. into assay (Fig. 1A–E), by counting the number of TH-IR neurons C57BL/6J mice for 6 consecutive days. Experimental mice (eight mice per http://www.jimmunol.org/ (Fig. 1F), or by morphological analysis of TH-IR neurons group) were also given either saline or doses of salmeterol at 1 or 10 mg/ (Fig. 1G). The 3H-DA uptake assay showed that salbutermol (the kg/day by continuous infusion (via an Alzet mini-pump) for 2 wk starting short-acting b2AR agonist) can protect DA neurons only at con- 2 d before the first MPTP injection. We then assessed the survival of DA centrations of 1027 and 1028 M (Fig. 1A), whereas long-acting neurons in the SNpc 21 d after the last MPTP injection through quantifi- cation of TH-positive neurons (A). Representative pictures of TH-IR neu- b2AR agonists clenbuterol, bambuterol, formoterol, and salme- rons staining for different treatment groups are shown (B). *p , 0.05, com- terol can protect DA neurons from LPS-induced toxicity at con- pared with the LPS-treated mice; #p , 0.05, compared with saline-treated 27 210 centrations ranging from 10 down to 10 M (Fig. 1B–E). mice. Scale bar, 50 mm. These results demonstrate that the neuroprotective effects of short- by guest on September 29, 2021 acting b2AR are ∼100- to 1000-fold less potent than long-acting compounds. Because of the slightly higher potency of salmeterol paper. Similar protective effects on DA neurons were observed against LPS-induced DA neurotoxicity when compared with the with salmeterol when we quantified the number of TH-IR neurons other long-acting agonists (Fig. 1E), and its wide usage as a clin- remaining in culture after 7 d with immunostaining (Fig. 1F). ical treatment for asthma and COPD, we used salmeterol as our Morphological analysis revealed that LPS treatment not only de- prototype long-acting b2AR agonist for further studies in this creased the number of TH-IR neurons but also caused a loss of neuronal processes, and these characteristics were also reversed by low doses of salmeterol pretreatment in a concentration-dependent manner (Fig. 1G). In addition to the pretreatment studies, a post-treatment of salmeterol was performed. Salmeterol at 10210 M was added to neuron-glia cultures 30 min prior, simultaneously with, or 30 min, 1 h, 2 h, and 3 h following the addition of LPS, and DA-neu- rotoxicity was measured 7 d later. We chose the 10210 M con- centration of salmeterol for these experiments because this was the lowest consistently effective concentration of salmeterol and other long-acting agonists that provided optimal neuroprotection in our pretreatment studies. The results in Fig. 2 showed that when salmeterol was added up to 2 h after LPS treatment, significant neuroprotection of TH-IR neurons was observed. We also ob- served that the production of inflammatory mediators was in- FIGURE 3. Salmeterol-mediated neuroprotection is microglia depen- hibited by the addition of salmeterol up to 2 h post-LPS treatment dent. Mesencephalic midbrain neuron-glia cultures (open bars, NG, which (data not shown). However, when salmeterol was added 3 h post- contain ∼10% microglia), neuron-enriched cultures (solid bars, N, which LPS treatment, no significant neuroprotection or inhibition of in- , contain 0.1% microglia) and neuron-astrocyte cultures (cross-hatched flammatory mediator production was observed. These results bars, NA, which contain ,0.1% microglia) were pretreated with either 2 demonstrate that the addition of salmeterol within the first few vehicle or 10 10 M salmeterol for 30 min prior to the addition of 0.2 mM MPP+. The 3H-DA uptake measurements were performed 7 d following hours following exposure to LPS is critical in determining the MPP+ treatment. Results were expressed as a percentage of the vehicle- survival of DA neurons. treated control cultures and were the means 6 SE from three independent The neuroprotective effect of salmeterol was further studied + experiments in triplicate. *p , 0.05 compared with the MPP+-treated using MPP , the active metabolite of MPTP, as the initiator of DA cultures. neurotoxicity in neuron-glia cultures (34). MPP+ is known to kill 6 SALMETEROL-MEDIATED NEUROPROTECTION

Table I. Striatal levels (ng/100 mg wet sample) of DA and its metabolites at 21 d after the last MPTP injection

DA (mean 6 SE) DOPAC (mean 6 SE) % (DOPAC/DA) HVA (mean 6 SE) % (HVA/DA) Saline control 748.5 6 76.7 123.0 6 14.3 16.4 107.1 6 4.26 14.3 MPTP 266.0 6 22.18## 81.33 6 10.2## 30.5## 73.11 6 5.66# 27.4## Salmeterol (10 mg/kg/day) 668.6 6 67.5 193.3 6 28.0## 28.8## 101.9 6 7.81 16.5 MPTP + salmetrol (1 mg/kg/day) 214.1 6 16.89 148.2 6 9.7** 69.2** 85.68 6 2.68* 39.7** MPTP + salmeterol (10 mg/kg/day) 205.6 6 16.23 165.9 6 8.8** 80.5** 97.18 6 5.77* 47.3** Mice were injected with vehicle or MPTP as described in Materials and Methods. Mice were sacrificed, and striatal tissues were removed 21 d after the last MPTP injection. The levels of DA and its metabolites DOPAC and HVA were determined with HPLC and expressed as means 6 SEM. Six to 10 animals were used in each group. The ratio of DOPAC levels to DA levels (DOPAC/DA) or HVA levels to DA levels (HVA/DA) was used to measure the activity of DA neurons. Results were expressed as mean 6 SE from 6 to 10 mice in each group measured individually. #p , 0.05, ##p , 0.01 compared with saline-injected control mice; *p , 0.05, **p , 0.01 compared with MPTP-injected mice.

DA neurons directly, and our previous work has shown that neu- turnover rates than those of either the saline- or MPTP-treated ronal death induced by MPP+ is the result of both direct cytotoxic groups. These results suggest that salemeterol, because of its pro- effects on DA neurons by MPP+, as well as by reactive micro- tective effect on cell bodies of DA neurons in the SN, was able to gliosis induced by toxic factors released by dying neurons (11, preserve and maintain higher activity of DA neurons. Therefore, 34). Treatment with 0.2 mM MPP+ for 7 d resulted in a 74% similar to our previous report (30), it is highly likely that as long decrease in DA neuron function in neuron-glia cultures, and pre- as the nigral DA neuronal bodies are preserved by the neuro- Downloaded from treatment with 10210 M salmeterol restored significant but not protective agents, the DA levels eventually will return to normal complete DA neuronal function in these cultures (Fig. 3). To values. further determine how salmeterol protects DA neurons from the A second PD model is the chronic inflammatory-based LPS cytotoxic effects of MPP+, we treated cultures of rat midbrain model. Our previous observations have shown that a single sys- lacking all glial cells (neuron-enriched cultures) or cultures con- temic injection of LPS elicits a time-dependent inflammatory- taining only neurons and astrocytes but not microglia (neuron- mediated progressive loss of DA neurons in the SN and a con- http://www.jimmunol.org/ astrocyte cultures) and measured DA neurons function 7 d after comitant loss of motor activity (28, 29). Three months after a treatment. Our results show that salmeterol was not capable of single injection of LPS (5 mg/kg, i.p.), the mid-time point between protecting DA neurons from MPP+-induced toxicity in either the this LPS injection and any observable DA-neuronal degeneration, neuron-enriched cultures or the neuron-astrocyte cultures (Fig. 3). C57BL/6J mice were infused with either saline alone or salmeterol These results demonstrate that microglia, not neurons or astroglia, given at two different doses (1 or 10 mg/kg/day) for a period of 2 serve as the target of a salmeterol-mediated effect against MPP+ wk via an Alzet mini-pump. Eight months after the LPS injection, -induced toxicity and might function by inhibiting the reactive rotarod behavioral tests were performed and again at 10 mo when microgliosis or by mediating direct neuroprotection elicited from mice were killed for histological analysis (Fig. 5A). Treatment MPP+-induced DA neuronal toxicity. with salmeterol at 10 mg/kg/day significantly protected the LPS- by guest on September 29, 2021 treated mice from motor deficits measured by rotarod activity at Salmeterol significant attenuates the loss of DA neurons in both 8 and 10 mo, whereas the 1 mg/kg/day dose showed signif- SNpc induced by systemic injection MPTP or LPS in vivo icant protection at 8 mo but not 10 mo (Fig. 5B). We further Because low doses of salmeterol shows potent DA-neuroprotective observed that salmeterol treatment significantly prevented LPS- effects in vitro, two different animal PD models were used for the induced loss of cell bodies and processes of DA neurons within substantiation of in vivo efficacy of salmeterol in preventing DA the SNpc at 10 mo (Fig. 5C,5D). Taken together, our data showed neurodegeneration. The first one is the MPTP model, initially used that a salmeterol treatment significantly attenuates the loss of DA in this study to validate the therapeutic efficacy of salmeterol (30). neurons in the SNpc induced by the injection of either MPTP or Mice received daily injections of MPTP (15 mg/kg, s.c.) for 6 LPS in vivo. consecutive days. Experimental mice were also given either saline or two different doses of salmeterol (1 or 10 mg/kg/day) by con- Salmeterol inhibits the LPS-induced increase in the production tinuous infusion (via an Alzet mini-pump) for 2 wk starting 2 of microglial proinflammatory factors d before the first MPTP injection. Three weeks following the last Activation of microglia either through the direct effects of LPS MPTP injection, mice were euthanized for morphologic analysis. on microglia or by reactive microgliosis via the death of neu- MPTP-injected mice displayed a 50% loss of TH-positive neurons rons produces an array of proinflammatory mediators, including (DA neurons) in SNpc compared with saline-injected controls, and ROS, TNF-a, and NO, which are the pivotal products that me- this decrease was significantly prevented by treatment with both diate inflammation-mediated neurotoxicity (11, 35). To determine doses of salmeterol (Fig. 4A). In addition, the loss of DA neurites whether salmeterol affects the generation of inflammatory medi- in the pars reticulata region of SN by injection of MPTP was also ators, primary microglia were pretreated with salmeterol before fully protected by salmeterol treatment (Fig. 4). In addition to the the addition of LPS. At concentrations from 1027 to 10210 M analysis of DA neurons in the SN, levels of DA and its metabolites but not 10211 and 10212 M, salmeterol significantly reduced LPS- DOPAC and HVA were measured in the striatum, the region mediated superoxide production (Fig. 6A), iROS production (Fig. containing DA nerve terminals projected from the SN. Six con- 6B), TNF-a (Fig. 6C), and NO (Fig. 6D), respectively. The in- secutive daily MPTP injections reduced the DA level by ∼65% hibition of LPS-induced production of proinflammatory factors compared with saline injected group (Table I). Although salme- by salmeterol was not due to lower numbers of microglia, because terol treatment did not prevent the loss of DA levels caused by microglial cell counts based on Iba-1 immunostaining showed that MPTP, interesting results were observed with the turnover rate of salemterol had no effect on the absolute number of microglia over DA (expressed by the ratio of DA and its metabolites), which the 7-d culture period (data not shown). These results show that reflect the functional status of the DA release. Both doses of low-dose salmeterol can specifically inhibit LPS-induced activa- salmeterol treatment (1 and 10 mg/kg/day) showed much higher tion and proinflammatory mediator production by microglia but The Journal of Immunology 7

FIGURE 5. Salmeterol significantly attenuates the loss of rotarod activity and DA neurons in SNpc in- duced by systemic LPS injection. C57BL/6J mice re- ceived a single LPS injection (5 mg/kg, i.p.), and 3 mo later, mice were infused with either vehicle alone or salmeterol (1 or 10 mg/kg/day) for 2 wk (via an Alzet Downloaded from mini-pump). Eight months and 10 mo after LPS in- jection, rotarod tests were performed (A, B), and then at 10 mo, mice were sacrificed, brains were harvested and sectioned, and 24 sections (rostral to caudal, 4.52– 5.36 mm posterior to bregma) were collected as de- scribed in Materials and Methods. Eight evenly spaced brain sections from vehicle, LPS-injected, or LPS- http://www.jimmunol.org/ injected salmeterol-treated animals were immunos- tained with anti-TH Ab, and the number of TH-IR neurons in the SN was counted. Data were expressed as percent loss compared with saline-injected controls (C). Representative pictures of TH-IR neurons staining for different treatment groups are shown (D). *p , 0.05, compared with the LPS-treated mice; #p , 0.05, compared with saline-treated mice. Scale bar, 50 mm. by guest on September 29, 2021

had no effect on microglia viability. Interestingly, other long- microglial cells. For this purpose, TNF-a production was used acting b2AR agonists including bambuterol, fomoterol, and as the prototype of the inflammatory response induced by LPS. clenbuterol also show the similar pattern in the inhibition of the We first measured TNF-a production from microglial cultures production of the proinflammatory mediators TNF-a and NO from generated from b2AR-deficient or wild-type mice to determine microglia, whereas short-acting agonist salbutermol failed to in- whether low-dose salmeterol inhibition was b2AR dependent. hibit TNF-a and NO production at 1029 M (Supplemental Fig. 1). Results show that although salmeterol displayed a concentration- dependent inhibition of TNF-a production in wild-type mice (Fig. b The anti-inflammatory effects of low dose salmeterol are 2AR 7A), this b2AR agonist completely lost its inhibitory effect on b and -arrestin2 dependent but cAMP independent TNF-a production from the b2AR-deficient microglia (Fig. 7B). Previous reports have shown that anti-inflammatory responses of b2AR activation normally leads to intracellular production of higher concentrations of b2AR agonists (between 10 nM and 10 cAMP, resulting in the activation of the catalytic subunit of PKA mM) are mediated through the activation of the b2AR and its (37). Surprisingly, enriched primary microglia from wild-type induction of cAMP (2, 4, 5, 36). Because much lower concen- mice did not produce detectable cAMP after treatment with sal- trations of b2AR agonists were examined in this study, a series of meterol at concentrations 10210 M or lower, whereas microglia experiments were conducted aimed at elucidating the signaling treated with doses of 1028 Mor1029 M salmeterol produced pathway underlying the anti-inflammatory effect of salmeterol on detectable cAMP after treatment (Fig. 7C). Furthermore, we found 8 SALMETEROL-MEDIATED NEUROPROTECTION Downloaded from

FIGURE 6. Effect of salmeterol on LPS-induced production of proinflammatory factors from microglia. Microglia-enriched cultures were seeded at a density of 1 3 105/well. Cells were pretreated with vehicle or various concentrations of salmeterol for 30 min, followed by the addition of LPS. The

production of LPS-induced extracellular superoxide production (A) was measured as SOD-inhabitable reduction of WST-1. LPS-induced iROS was de- http://www.jimmunol.org/ termined by probe DCFH-DA (B). Salmererol’s effect on LPS-induced production of TNF-a and nitrite were shown in C and D. Results were expressed as mean 6 SE from three to five independent experiments in triplicate. *p , 0.05, **p , 0.01, compared with the LPS-treated cultures. that the PKA inhibitors Rp-cAMP and H89 failed to abolish the hibitory effect on the major LPS-induced proinflammatory path- inhibitory effects of low-dose salmeterol (10210 M) on the LPS- ways, namely MAPK and NF-kB (Fig. 8). Primary microglia cells induced production of TNF-a (Fig. 7D) or nitrite (Supplemental were pretreated with salmeterol at concentrations ranging from Fig. 2A) in microglia. In addition to the stimulation of PKA, other 1029 to 10212 M, then followed by stimulation with LPS for 15 effectors can also be activated by cAMP signaling including a min. ERK1/2, p38, and JNK were all phosphorylated in microglia small family of guanine nucleotide exchange factors also known following LPS stimulation (Fig. 8A), and the phosphorylation of by guest on September 29, 2021 as an exchange protein directly activated by cAMP (EPAC) (38). ERK1/2, p38, and JNK were all significantly reduced by pre- Furthermore, pretreatment with an EPAC agonist, 8CPT-29-O-Me- treatment with salmeterol at concentrations of 1029–10210 Mbut cAMP, did not inhibit the LPS-induced production of TNF-a (Fig. not 10212 M. Salmeterol at 10210 M also reduced the phosphor- 7E) or nitrite (Supplemental Fig. 2B), and it did not significantly ylation of p65–NF-kB (Fig. 8B) and the LPS-induced NF-kB alter the salmeterol-mediated inhibition of microglial activation, nuclear translocation and binding to the kB site (Fig. 8C). Iden- indicating EPAC activation does not play a role in the salmeterol- tical results were seen when TNF-a rather than LPS was used to mediated anti-inflammatory response. Because low-dose salme- activate microglial cells [i.e., NF-kB nuclear translocation and terol did not appear to activate the canonical b2AR/cAMP sig- kB-binding were inhibited by low-dose salmeterol (Fig. 8D)], naling pathway, we then investigated whether salmeterol’s effects suggesting this inhibition was not specific for the TLR4-mediated were mediated through b-arrestin2, a known negative regulator of pathway. Because TAK1 is the converging target for LPS- and inflammatory responses in monocytes and macrophages (39, 40). TNF-a–elicited activation of both the MAPK and NF-kB path- To test this possibility, primary microglia were transfected with ways, we investigated whether TAK1 phosphorylation induced by control or b-arrestin2–specific siRNA, treated with the indicated LPS stimulation of microglial cells was inhibited by salmeterol. concentrations of salmeterol, then exposed to LPS. We found that The results show that salmeterol at concentrations of 1029 and the anti-inflammatory effects of salmeterol were abolished by the 10210 M inhibited the LPS-induced phosphorylation of TAK1 siRNA-mediated knockdown of b-arrestin2 but not in the control (Fig. 8E,8F), suggesting that low-dose salmeterol-mediated po- siRNA-treated cells (Fig. 7F). Western blot analysis demonstrated tent anti-inflammatory effects are through the inhibition of TAK1- the efficiency of siRNA knockdown (Fig. 7G). Taken together, mediated MAPK and NF-kB signaling pathways. these data strongly suggest that low-dose salmeterol-mediated anti-inflammatory effects are dependent on b2AR/b-arrestin2 but Discussion independent of the cAMP/PKA and cAMP/EPAC pathways. One of the most salient features in this report is the finding that long-acting b2AR agonists exhibit potent neuroprotective and Salmeterol significantly suppresses LPS-induced MAPK and anti-inflammatory effects at concentrations 100-fold less than have k NF- B activation through inhibition of TAK-1 phosphorylation been found previously to have specific biological activity. Using It has been previously shown that b2AR-dependent responses well-established LPS- and MPP+-mediated in vitro and in vivo PD mediated by agonists at micromolar concentrations exert their models, we demonstrated that b2AR agonists are protective against anti-inflammatory effect primarily through the inhibition of NF- inflammation-induced degeneration of DA containing neurons and kB. However, the target(s) of the anti-inflammatory activity we that this protection is mediated through the b2AR-mediated inhi- observed at doses of 10210 M salmeterol are not known. Thus, we bition of microglial activation and its proinflammatory factors investigated whether this low dose of salmeterol showed any in- production. Mechanistic studies demonstrate that this low-dose The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Low-dose salmeterol-mediated anti-inflammatory effects are b2AR/b-arrestin2 dependent but cAMP/PKA and cAMP/EPAC independent. Microglia culture prepared from C57BL/6 (A)orb2AR-deficient mice (B) were pretreated with vehicle or indicated concentrations of salmeterol for 30 min prior to the addition of LPS. Supernatant were collected at 3 h after LPS addition for TNF-a analysis (A, B). C, Enriched primary microglia were incubated with vehicle or indicated concentrations of salmeterol at 37˚C for 5, 10, or 30 min. After incubation, the cells were lysed, and cAMP levels were determined using a cAMP assay kit. Data were expressed as picomoles cAMP per 1 million cells (C). D and E, Enriched primary microglia cells were pretreated with vehicle or PKA inhibitors, including H89 (1 mM for 45 min), Rp-cAMP (50 mM for 45 min) (D), or EPAC agonist 8CPT-29-O-Me-cAMP (CPTOMe) (10 mM for 45 min) (E) prior to stimulation with salmeterol (10210 M) and LPS (5 ng/ml). Supernatants were collected 3 h after LPS addition to measure TNF- a levels. F and G, Primary microglia cells were transfected with 100 pmol specific b-arrestin2 siRNA or control siRNA, and 48 h after transfection, cells were treated with indicated concentrations of salmeterol for 30 min prior to addition of LPS. Supernatants were collected for TNF-a assay (F). Knockdown of expression of the b-arrestin2 was determined by Western blot analysis (G). Results in A–F were expressed as mean 6 SE from three to four independent experiments in triplicate. *p , 0.05, **p , 0.01, compared with the LPS-treated cultures. salmeterol-mediated potent anti-inflammatory effect is medi- signaling pathways; rather, this inhibition is dependent on the ated through the inhibition of the phosphorylation of MAPK and expression of b-arrestin2, which suggests a novel mode of ac- p65 NF-kB in activated microglia and function independent tion for the long-acting b2ΑR agonists in regulating CNS in- of the conventional GPCR/cAMP/PKA or GPCR/cAMP/EPAC flammatory conditions. 10 SALMETEROL-MEDIATED NEUROPROTECTION Downloaded from http://www.jimmunol.org/

FIGURE 8. Salmeterol significantly suppresses LPS-induced MAPK and NF-kB activation through inhibition of TAK-1 phosphorylation. Enriched by guest on September 29, 2021 microglia were pretreated with vehicle or salmeterol (1029–10212 M) for 30 min, followed by treatment with LPS (10 ng/ml) for 15 min. Cells were then harvested, and the amounts of phosphorylated and total MAPK (ERK1/2, p38, and JNK1/2) (A) and phosphorylated and total NF-kB p65 (B) were de- termined by Western blot analysis, respectively. Representative Western blots for ERK1/2, p38, JNK, and p65 phosphorylation are shown from three independent experiments. C and D, Primary microglia were pretreated with salmeterol (1029–10212 M) for 30 min and then treated with LPS (C) or TNF-a (D) for 30 min. Nuclear extracts were prepared from these cells, and NF-kB (p65) DNA binding activity was detected using the NF-kB (p65) Transcription Factor Assay Kit. E and F, Enriched microglia were pretreated with vehicle or salmeterol (1029–10210 M) for 30 min, followed by the treatment with LPS (10 ng/ml) for 15 min, cells were then harvested, and the amounts of phosphorylated TAK1 and total TAK1 are show in Western blot analysis using specific Abs (E). ImageJ software was used to quantitate the intensity of the phosphorylated TAK1 and total TAK1 bands in Western blot, and the results given in F represent the percentage difference of the ratio of phosphorylated TAK1 compared with total TAK1 normalized to the vehicle-treated control (F).

Long-acting b2AR agonists including salmeterol are now cur- “reactive microgliosis,” which causes additional neurodegenera- rently in use for the treatment of chronic pulmonary diseases such tion (41). Thus, between microglia activation and neuronal death, as asthma and COPD (8, 9). Our data proposes a new indication a vicious, self-propelling cycle is formed and drives progressive for this class of compounds in the treatment of chronic neurode- neurodegeneration (34). It is interesting to note that 3 mo after generative diseases such as PD. This possibility is based on the LPS injection, a 2-wk treatment with extremely low dose of sal- efficacy of salmeterol shown in two different in vivo PD models, meterol (1–10 mg/kg/day) is sufficient to achieve neuroprotection a subacute neurotoxin MPTP model and a chronic inflammatory and improvement of motor deficits. This finding lends further cre- LPS model. Interestingly, our results showed that salmeterol dis- dence to the notion that breaking the vicious cycle of inflammation- plays protective effects in both of these PD models, even though mediated neurodegeneration in the diseased brain is a new and effi- the mode of action of these two toxins is different. In the LPS cient therapy for the treatment of PD and perhaps other long-term model, we have previously reported that a single systemic in- chronic neurodegenerative diseases (34). jection produces chronic neuroinflammation, which results in Equally potent efficacy of salmeterol’s action in protecting delayed, progressive motor deficits and selective loss of nigral DA against DA neurodegeneration was also observed in MPTP model. neurons over a period of 7–10 mo (28, 29). Our in vitro studies It is interesting to note that MPTP initially exerts a specific neu- have shown that the most likely mechanism of this chronic LPS- rotoxic effect on DA neurons, and our studies show that salmeterol mediated neurodegeneration is through the activation of microglia, has no direct protective effect on DA neurons against MPP+- which results in the acute production of inflammatory mediators. mediated toxicity (Fig. 3). This finding, and our previous work This initial acute inflammatory response results in the genera- (42), suggests that a significant amount of neurodegeneration seen tion of dying or damaged neurons, which in turn release toxic in the MPTP model is mediated by microglia-mediated inflam- substances to further activate microglia through a process called matory killing, and one possible mechanism underlying salmeterol- The Journal of Immunology 11 elicited protection is through inhibition of reactive microgliosis. dose salmeterol can be potently but selectively anti-inflammatory in This observation is consistent with previous results both from our microglial cells by targeting the MAPK and NF-kB signaling laboratory and from others using anti-inflammatory therapies such pathways following LPS activation. as NF-kB inhibitors (43), regulatory T cells (44, 45), or anti- The neuroprotective effects of b2AR activation by higher doses inflammatory cytokines (46) to prevent MPTP-mediated neuro- of b2AR agonists have been reported in other conditions such degeneration. In summary, both our in vivo and in vitro results as amyotrophic lateral sclerosis (52), cerebral ischemic (53), and clearly demonstrate that, despite the difference in the mode of spinal cord injury-induced locomotor dysfunction (54). Although action, salmeterol displays potent neuroprotection in both LPS and it is not yet clear how the b2AR agonists exhibit all these neu- MPTP-induced PD models. On the basis of these findings, we roprotective properties, several studies suggested that b2AR propose that the anti-inflammatory effect of low-dose long-acting agonists function to stimulate glutathione-dependent antioxidant b2AR agonists is capable of inhibiting both MPTP-induced re- processes from nerve cells (54), and others reported that neuro- active microgliosis and LPS-induced acute microglial activation trophic factors from activated astocytes induced by b2AR agonists and ultimately suppress neuroinflammation that mediates chronic contribute to neuroprotection (55, 56). We propose that the major neurodegeneration in PD. neuroprotective activity of b2AR agonists in this study was due to Increasing evidence from both in vivo and in vitro studies suggest their anti-inflammatory properties. Our results clearly show that that b2AR agonists possess important immunomodulatory po- the effectiveness of the b2AR agonist salmeterol and the other tential. Our studies provide a novel pathway that is different from long-acting b2AR agonists at the low concentrations tested in this the conventional cAMP/PKA/NF-kB–dependent pathway medi- paper is due to their anti-inflammatory effect on microglia and ated by b2AR activation. These findings also provide opportuni- not to a direct protective effect on DA-neurons or through an Downloaded from ties for novel therapeutic approaches in inflammation-mediated astrocyte-dependent effect (Fig. 3). Given the effectiveness of CNS disorders such as PD. Although bronchodilation occurs via these compounds at such low concentrations in inhibiting inflam- the classical b2AR/cAMP/PKA pathway (47), it is clear that low matory responses, they appear to have significant potential in regu- dose salmeterol-mediated anti-inflammatory and neuroprotective lating CNS inflammation and the treatment of chronic inflammatory effects are not PKA or EPAC dependent because PKA inhibitors disorders of CNS.

could not block the anti-inflammatory activity of salmeterol, and http://www.jimmunol.org/ EPAC agonists could not mimic their anti-inflammatory effect Acknowledgments (Fig. 7D,7E). For this reason, we looked for an alternative path- We thank Drs. Robert Langenbach and Robert Oakley at the National In- way mediating salmeterol-related protective effects. Arrestins, stitute on Environmental Health Sciences for helpful suggestions for this originally discovered as terminators of GPCR signaling by facil- paper. itating desensitization and internalization of b2AR, recently have been recognized as multifunctional adaptor/scaffold proteins in Disclosures regulating cellular processes such as chemotaxis, apoptosis, me- The authors have no financial conflicts of interest. tastasis, and inflammation (40, 48, 49). b-Arrestin2 has been found to be a negative regulator of inflammatory responses in by guest on September 29, 2021 monocytes and macrophages (39, 40), but its role in microglia References remains unknown. Our studies demonstrate that low doses of 1. Koff, W. C., A. V. Fann, M. A. Dunegan, and L. B. Lachman. 1986. Catecholamine-induced suppression of interleukin-1 production. 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