Introduction

Parkinson’s disease (PD) is a complex, progressive, neurodegenerative disorder characterized by SNc DA neuron (DAN) death1,2. PD prevalence is higher in males and PD symptoms often go unnoticed until >75% of SNc neurons have died3,4. Phenotypically, PD patients exhibit profound motor impairment that includes bradykinesia, resting tremor, muscular rigidity, lack of coordination, and postural instability5. These symptoms are due to SNc DAN cell death and concomitant diminished striatal DA signaling, and PD therapeutic strategies typically aim to boost DA production in the remaining DAN population6.

Rit2 (AKA: Rin, Ras-like in neurons) is a small, neuronal, ras-like GTPase with enriched expression in SNc DANs7. Rit2 directly interacts with the DA transporter (DAT), and is required for regulated DAT membrane trafficking8,9,10. In cell culture models, Rit2 is required for EGF- and NGF-mediated neurite outgrowth, NGF-mediated ERK phosphorylation, and cell viability11,12,13,14. Genome-wide association studies (GWAS) link Rit2 genetic anomalies to PD15,16,17,18,19,20,21,57. Gait testing apparatus consisted of a 10 cm x 36 cm runway with 14 cm high foamboard walls and a dark box at the opposing end. Fresh, legal-size paper was placed on the benchtop under the runway for each trial. Mouse forepaws and hind-paws were dipped in non-toxic orange and blue tempera paint, respectively, and mice were placed on the paper at the open end of the runway and allowed to traverse to the closed box at the opposing end. Three trials were performed per mouse and stride length, stride width and tow spread were measured for both fore- and hindlimbs. Number of completed trials was also quantified. Experimenters and data analysts were double-blinded to mouse IDs.

Grip strength

Four-limb grip strength was measured using the Bioseb Grip Strength Test (BIO-GS3) equipped with mesh grip grid for mice. Mice were suspended by tail over the mesh and lowered onto it until all 4 paws grasped the mesh. The mouse was then pulled backwards gently on the horizontal plane until it released from the mesh. The maximal force applied was recorded for 3 consecutive trials and averaged.

Tissue harvesting and immunoblotting

Striata were collected by preparing 300 µm coronal sections on a Vibratome as previously described8,9. Sections were collected through the entire striatum, dorsal and ventral striata were sub-dissected, and slices encompassing each region were pooled for each independent mouse. Tissue was lysed in RIPA buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 1.0 mM EDTA; 0.1% SDS, 1% Triton X-100, 1% Na deoxycholate) supplemented with protease inhibitors (1.0 mM phenylmethylsulfonyl fluoride and 1.0 g/mL each leupeptin, aprotinin, and pepstatin) and Phosphatase inhibitor cocktail V (EMD Millipore). Mechanical tissue disruption was also performed by triturating sequentially through a 200 µL pipette tip, 22-, and 26- gauge tech tips and solubilized by rotating (30 min 4°C). Insoluble material was removed by centrifugation (15 min, 18 K x g, 4°C). Lysate protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific). Protein samples were denatured in an equal volume of 2x Laemmli sample buffer and were either rotated (30 min, RT) for membrane protein immunoblots or boiled (5 min) for soluble protein immunoblots. Proteins were resolved by SDS-Page, transferred to nitrocellulose membranes, and the indicated proteins were detected and quantified by immunoblotting with the following antibodies: rat anti-DAT (MAB369, Millipore; 1:2000), rabbit anti-TH (AB152, Millipore, 1:10000), rabbit anti-pSer40 TH (AB5935, Millipore, 1:5000), rabbit anti-αSyn, rabbit anti-pSer129-αSyn, anti-LRRK2, anti-pSer935 LRRK2, mouse anti-actin (Santa Cruz, 1:5000). Secondary antibodies conjugated to horseradish peroxidase were all from Jackson ImmunoResearch and immunoreactive bands were visualized by chemiluminescence using SuperSignal West Dura (Thermo Scientific). Immunoblotting solutions were prepared in either PBS-T, or TBS-T (137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, ph7.4, 0.1% Tween20) when probing for phosphoproteins. Non-saturating immunoreactive bands were detected using either VersaDoc 5000MP or Chemidoc imaging stations (Bio-Rad) and were quantified using Quantity One software (Bio-Rad). Representative blots shown for a given condition were cropped from the same exposure of the same immunoblot and spliced together for presentation purposes only. Splice margins are indicated with a line. All compared blots were processed in parallel and derive from the same experiments.

Fast-scan cyclic voltammetry

Mice were sacrificed by cervical dislocation and rapid decapitation. Heads were immediately submerged in ice-cold NMDG cutting solution, pH 7.3-7.4 (20 mM HEPES, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2·4H2O. 10 mM MgSO4·7H2O, 92mM N-methyl-D-glucamine, 2 mM thiourea, 5 mM Na+-ascorbate, 3 mM Na+-pyruvate). Brains were removed, glued to the stage of a VT1200S Vibroslicer (Leica) and submerged in ice-cold, oxygenated cutting solution. 300 µm slices were prepared and were hemisected along the midline prior to recovering in ACSF (125 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 26 mM NaHCO3, 11 mM glucose, 2.4 mM CaCl2·4H2O,1.2 mM MgCl2·6H2O, pH 7.4) at 31°C for a minimum of 1 hour prior to recording. Hemislices were moved to the recording chamber and were perfused with oxygenated ASCF supplemented with 500 µM Na-Ascorbate. Glass pipettes containing a 7 µm carbon-fiber microelectrode were prepared and preconditioned in ASCF by applying triangular voltage ramps ( − 0.4 to +1.2 and back to −0.4 V at 400 V/s), delivered at 60 Hz for 1 hour. Recordings were performed at 10 Hz. Electrodes were calibrated to a 1 µM DA standard prior to recording. Electrodes were positioned in DS and DA transients were electrically evoked with a 250 µA rectangular pulse every 2 min, using a concentric bipolar electrode placed ~100 µm from the carbon fiber electrode. Data were collected with a 3-electrode headstage, using an EPC10 amplifier (Heka) after low-pass filter at 10 kHz and digitized at 100 kHz, using Patchmaster software (Heka). A stable baseline was achieved after evoking six consecutive DA transients, after which experimental data were collected. Each biological replicate is the average of three evoked DA transients/slice, and a minimum of 3 independent mice were used to gather data from the indicated number of slices in each experiment. Data were analyzed in Igor Pro, using the Wavemetrics FSCV plugin (gift of Veronica Alvarez, NIAAA). Peak amplitudes were measured for each individual DA transient, and tau was calculated as 1/e according to the equation: y = y0 + A((x-x0)/tau)).

Mass spectrometry

Sample preparation

Brains were harvested, 1.0 mm coronal sections were prepared and bilateral 1.0mm2 punches were each taken from dorsal and ventral striata. Each bilateral pair was solubilized in 10 µL internal standard solution (200 µM 13C4-GABA and 1 µM 2H3-DA in water with 500 µM ascorbic acid and 0.1% formic acid) and 50 µl ice-cold acetonitrile with 1% formic acid. Samples were vortexed twice for 0.5 min with a 1 min incubation on ice between vortexing and were sonicated in an ice-water bath until tissue was completely disrupted. Samples were centrifuged (10 min, 16,000 x g) and supernatants were collected for LC/MS/MS analysis. A standard (STD) solution containing 200 µM GABA, 1 µM dopamine, 500 µM ascorbic acid and 0.1% formic acid was also prepared.

LC/MS/MS

10 µl samples were injected in triplicate into a Thermo Scientific Ultimate 3000 HPLC system on a SeQuant ZIC-cHILIC column (2.1 ×100 mm, 3 µm) with a ZIC-cHILIC guard column (2.1 ×20 mm, 5 µm), coupled with a Thermo Scientific TSQ Quantiva triple quadrupole mass spectrometer. The mobile phase was water with 0.1% formic acid (A) and acetonitrile (B), and the elution program was as follows: 0 min 25% A, 0.5 min 25% A, 4.5 min 45% A, 5.0 min 70% A, 8.0 min 70% A, 8.1 min 25% A, 12.0 min 25% A at 0.2 mL/min. Ionization was operated in the positive mode with the voltage of 4.2 kV. The parameters were set as follow: sheath gas, 35 Arb, aux gas, 15 Arb, vaporizer temperature, 250 °C, ion transfer tube temperature, 325 °C. Multiple reaction monitoring (MRM) was performed using a cycle time of 0.3 s, CID gas pressure of 1.5 mTorr, Q1 resolution (FWHM) of 0.7 and Q3 resolution (FWHM) of 0.7. The MRM transitions 104.1 > 87 (GABA), 108.1 > 91 (13C4-GABA), 154.1 > 91 (dopamine) and 157.1 > 93 (2H3-dopamine) were selected for quantification. All data was integrated and processed in Xcalibur (Version 2.2, Thermo Scientific).

Stereological analysis and Immunohistochemistry/confocal microscopy

Briefly, mice were perfused and fixed with freshly made 4% paraformaldehyde (PFA) in PBS. Brains were removed immediately and fixed again in 4% PFA followed by equilibration in 30% sucrose in PBS. Midbrains were removed for stereological analysis, and forebrains were used for immunohistochemistry/confocal microscopy.

Stereological analysis

SNc total and TH+ neurons were quantified as previously described58. Fixed brains were imbedded in the OCT-compound media (Sakura) and frozen in liquid nitrogen. 40 µm cryosections were prepared through the midbrain a Leica CM3050s cryostat, and were stored in an antifreeze media containing 30% ethylene glycol, 25% glycerol, and 5% phosphate buffer. For stereology counting, 1 in every 5 sections was selected with a random start and a total of 6 brain slices on average were used for each mouse for IHC labeling for TH, including DAB enhancement, followed by Cresyl violet staining to reveal all neurons. Substantia nigra pars compacta was imaged using a Zeiss Axioplan 2 microscope equipped with a 20X objective, and Stereo Investigator was used to estimate the total number of neurons in the region of interest using the following parameters: frame sizes, 150 ×150 µm; grid sizes, 250 ×250 µm; top guard zone height, 2 µm; and optical dissector height, 8 µm. These parameters yielded a coefficient of error <10% throughout the analysis. Total cell numbers measured were weighted to section thickness for each mouse and were averaged across each cohort. Investigators performing stereological counting were blinded to mouse identity.

Immunohistochemistry/confocal microscopy

25 µm coronal sections through the striatum were prepared using a microtome and were co-stained with rabbit anti-pSer129-Syn (Cell Signaling #23706; 1:500) and chicken anti-TH (Millipore #AB9702, 1:500), followed by staining with secondary antibodies preabsorbed against mouse (Alexa568-goat anti-rabbit, Alexa647-donkey anti-chicken, Jackson ImmunoResearch). Z-stacks in the dorsal striatum were acquired with a Zeiss 700 LSM scanning confocal microscope using 555 nm and 647 nm lasers, and were pseudocolored to red and green, respectively. Acquisition settings within each channel (pinhole size, digital gain, and laser strength) were identical across all samples. Z-stacks were imported into ImageJ software where a representative plane was chosen, channels were separated (for individual red and green images), and images exported as tiff files. Files were subsequently imported into Adobe Photoshop, and levels were adjusted identically across all images.

Statistics

Data analysis was performed with GraphPad Prism software. All data were assessed for normality and nonparametric tests were applied if data distribution was non-Gaussian. Outliers in each data set were identified using either Grubb’s or Rout’s outlier tests, with a or Q values set at 0.05 or 5%, respectively, and were removed from further analysis. Significant differences between two values were determined using either a one-tailed, two-tailed, or paired Student’s t test, as indicated. Differences amongst more than two conditions were determined using one-way or two-way ANOVA, as appropriate, and significant differences among individual values within the group were determined by post-hoc multiple comparison tests, as described for each experiment.