Animals
C57BL/6J mice (3–4 months old) were purchased from Jackson Laboratory (Bar Harbor, ME). Male mice were used due to ~80-100% MPTP mortality in female mice based on our observations. Mice were kept in a temperature-controlled room, with a 12-h light/dark cycle, and had free access to food and water. To assess the MC1R-dependence of effects, Mc1re/e mice (3–4 months old) were used. Mc1re/e mice carry an inactivating mutation of Mc1r in a C57BL/6J background. All procedures were approved by the Institutional Animal Ethical Committee of Massachusetts General Hospital (animal protocol # 2018N000039).
Chemicals and treatment paradigms
García-Domínguez et al. reported an enhanced microglia response and nigral dopaminergic cell death in an acute MPTP model following peripheral inflammation resulting from single i.p. injection of LPS at 2 mg/kg (43). We employed a subacute paradigm to inject lower doses of MPTP.HCl (20 mg/kg) and LPS (1 mg/kg) over 4 days to induce systemic inflammation. Mice were randomly divided into MPTP+LPS+NDP-MSH, MPTP+LPS, and control groups to receive i.p. once daily MPTP.HCl (Millipore Sigma, Cat# M0896; 20 mg/kg) or saline and LPS (Millipore Sigma, Cat# L4391; 1 mg/kg) or PBS from day 1 to day 4. NDP-MSH (Genscript, Cat# RP10658; 400 µg/kg) or PBS was injected from day 1 to day 12. Mice were tested for behavioral activities and were sacrificed thereafter on day 12.
To study the role of Tregs, animals were treated with anti-mouse CD25 monoclonal antibody (clone PC61, Biolegend, Cat# 102059; 400 µg/mouse) or isotype control (Biolegend, Cat# 401916; 100µg/mice) for 3 alternate days, 1 week before the start of experiment. Mice were subsequently treated with MPTP, LPS and NDP-MSH as described above. Another dose of anti-mouse CD25 monoclonal antibody or isotype control was administered 2 days before the sacrifice.
BBB permeability and NDP-MSH pharmacokinetics (PK) study
The integrity of BBB was measured through FITC-albumin (Millipore Sigma, Cat# A9771) leakage from vasculature into brain parenchyma as described previously (44). Mice were treated with MPTP+LPS and sacrificed after 6 h and 24 h after the last dose. Briefly, mice were anaesthetized by isoflurane and perfused intracardially with heparin (100 units/kg) followed by 5 ml FITC albumin at a concentration of 5 mg/ml in PBS with a flow rate of 1.5 ml/min. Subsequently, the brain was isolated and incubated in 4% paraformaldehyde overnight. The solution was changed to 30% sucrose in PBS. Coronal sections of striatum were mounted and analyzed under fluorescence microscope (Olympus BX51 microscope).
To assess NDP-MSH concentrations in plasma and brain, male mice were treated with MPTP+LPS as described above and two concentrations of NDP-MSH (400 µg/kg and 1 mg/kg) and sacrificed after 5-, 30- and 90-min. Blood samples were collected through cardiac puncture in 40 mM EDTA, and plasma was collected by centrifugation and stored at -80°C till further analysis. Whole brain was dissected and homogenized in PBS. Proteins in brain homogenate and plasma samples were crashed with 3 volumes of methanol containing internal standard (propranolol) and centrifuged. Supernatants were analyzed by liquid chromatography/mass spectrometry (LC/MS). NDP-MSH in plasma and brain samples was detected by LC/MS through a service contract with Cyprotex, LLC, MA, USA.
Open field test
Locomotor activity was determined at the baseline and post treatment by open field test. Briefly, the mice were placed in the plexiglass chamber (11 × 11 in with clear 8-in high walls) and were allowed to explore for a period of 10 min. The total distance travelled was measured with software Ethovision XT 9.0, Noldus Information Technology, The Netherlands.
Pole test
Pole test was performed at the baseline and post treatment to test motor coordination and motor abnormalities that result from depletion of striatal dopamine. Mice were trained on the pole (1 cm diameter, 50 cm height) one day before the start of the experiment for 120s. Time taken by the mice to turn (T turn) and time taken to climb down (T descent) the pole were recorded (45,46).
Immunohistochemistry and stereological counting of SN dopaminergic neurons
Immunohistochemistry was performed on the coronal sections of SN as described previously (12). In brief, the 30µm sections were incubated in blocking solution (10%, normal goat serum) for 1h followed by incubating them with either of the following primary antibodies (Enzo Life Sciences, Cat# BML-SA497-0100, Tyrosine Hydroxylase (TH) (1:1000); Abcam, Cat# ab178847, Ionized calcium binding adaptor molecule 1 (iba1) (1:500); Biolegend, Cat# 840001, Glial Fibrillary Acidic Protein (GFAP) (1:500) overnight at 4ºC. For peroxidase staining, sections were incubated with biotinylated secondary antibodies (Millipore, Cat# OS03B, anti-rabbit 1:2000); Sigma-Aldrich, Cat# B7264, anti-mouse 1:2000) followed by incubating in avidin biotin complex (Vector laboratories, Cat# PK6100) and the staining was developed by incubation in 3,3′-diaminobenzidine (DAB) (Millipore Sigma, Cat# D4418). TH, iba1, and GFAP are markers for dopaminergic neurons, microglia, and astrocytes, respectively.
Stereological counting of SN TH+ cells was performed to determine the total number of dopaminergic neurons in the SN as previously described (12). In brief, a complete set of coronal midbrain sections stained with TH and counterstained with Nissl was counted stereologically with Olympus BX51 microscope and Olympus CAST stereology software.
The method published by Sanchez-Guajardo et al. (2010) was referred to for analysis and classification of morphology of iba1+ microglia cells in SNpc (47). These cells can be classified according to their morphology into resting type (type A with a thin and visible cytoplasm with long and thin processes), activated type (type B with thick and short processes extending from a dense and enlarged cell body), and phagocytic type (type C with a shape resembling pseudo-amoeba, a big and dark cell body with processes). The stereological method was followed to count the cells at 40× magnification (Olympus BX51 microscope and Olympus CAST stereology software) as previously described by Dimant et al., 2013 and West et al., 1991(48,49). Two midbrain sections with the central and anterior SN were analyzed per mouse.
Integrated optical density of GFAP immunoreactivity was determined by Image J as a measurement of astrogliosis. The images were captured using ×40 objective. Two midbrain sections with the central and anterior SN were analyzed for each mouse. The general protocol used for TH cell staining was deposited in protocols.io (DOI: dx.doi.org/10.17504/protocols.io.j8nlk4yw1g5r/v1)
High-performance liquid chromatography
High-performance liquid chromatography with electrochemical detection (HPLC-ECD) was used to measure striatal dopamine levels as previously described (50,51). Briefly, the striatum was dissected from the brain, homogenized in buffer containing perchloric acid and centrifuged at 16000g for 20 min followed by analysis of the supernatant through HPLC-ECD. The general protocol used for measurement of dopamine was deposited in protocols.io (DOI: dx.doi.org/10.17504/protocols.io.dm6gpbjdplzp/v1).
Additionally striatal 1-methyl-4-phenylpyridinium (MPP+) was measured 90 min and 6 h after the last dose of MPTP. Striatum was dissected and analyzed by HPLC ultraviolet-ultraviolet (UV) detection as previously described (52).
ELISA
Levels of IL-1β and TNF-α in plasma and brain tissue were determined by ELISA as described previously (53). In brief, blood samples from all the treatment groups were collected through cardiac puncture in 40 mM EDTA and plasma was collected by centrifugation. The plasma samples were immediately transferred to dry ice and then stored at -80°C till further analysis. A small fraction of ventral midbrain tissue homogenate prepared in 1x RIPA buffer (Cell Signaling, Cat#9806) was used for analysis of IL-1β and TNF-α using mouse ELISA kits (Biolegend Cat# 430904; 432604).
Flow cytometry
To assess immune cell profile, single-cell suspension of spleen tissue was prepared according to the protocol described previously (54). Spleen was removed in a 35 mm petri plate with 5 ml RPMI 1640 and digested mechanically and passed through 70 μm filter screen. The cell suspension was centrifuged and the pellet was incubated in RBC lysis buffer. The resulting cell suspension was washed in 1xPBS and blocked with Fc Block (Biolegend, Cat# 101302, 1 μl/50 μl). The cells were incubated with MC1R (Invitrogen, Cat# PIPA521911, 1.39 µg) antibody followed by fluorophore conjugated primary antibodies for extracellular markers (Biolegend Cat# 101235, CD11b-BV421(0.25 µg); Cat# 127641, Ly6G-BV-650 (0.25 µg); Cat# 128041, Ly6C-BV785 (0.125 µg); Cat# 100516, CD4-APC (0.25 µg); Cat# 100751, CD8a-BV510 (0.5 µg); Cat# 152405, CD19-PerCP-Cy5.5 (0.25 µg); Cat# 102036, CD25-BV605 (0.3 µg)) and AF488 (Invitrogen, Cat# A11034, 1:200). Zombie dye (Biolegend, Cat# 423101, 1µl/sample) was used to differentiate between live and dead cells. Helper T cells and cytotoxic T cells were identified by CD4+ and CD8+, respectively. CD4+CD25+ cells were used to mark Tregs. CD19+ cells were used as marker for B cells. Monocytes were identified as CD11b+Ly6G-Ly6Chigh cells and neutrophils were marked by CD11b+Ly6C-Ly6G+. For monocytes and neutrophils, CD11b-positive cells were first extracted from the live cell subset by expansion with SSC-A, followed by Ly6C. After expansion with Ly6C and Ly6G, we excluded Ly6G-positive cells. Monocytes were identified as CD11b+Ly6G-Ly6Chigh cells, and neutrophils were marked by CD11b+Ly6C-Ly6G+. SORP 5 Laser BD Fortessa X-20 (BD Bioscience) and FlowJo v10.7.1 (Becton Dickson & Company) software was used for data acquisition. Single cell control and manual compensation was used for gating strategy. Abundance of the cell population was calculated and presented as % of the population relative to live cells. FlowJo v10.7.1 (Becton Dickson & Company) software was used for data analysis.
Statistical analysis
Data from each experiment was represented as mean ± SEM and statistical significance was determined by One-way ANOVA with Tukey post hoc test. Two-way ANOVA with Tukey post hoc test was used to analyze neurobehavioral endpoints in open field test and pole test to compare baseline and post treatment effects. GraphPad Prism 8.3.0 (GraphPad Software, San Diego, CA, USA) was used to analyze the data.