Background

α-synuclein (α-syn) is a major component of Lewy bodies (LB) and Lewy neurites (LN) appearing in the postmortem brain of Parkinson's disease (PD) and other synucleinopathies [1, 2]. Genetic mutations in α-syn, including point mutations (A53T, A30P and E46K) and multiplications have been linked to familial PD and Dementia with LB [3–7]. Although the precise function of α-syn remains elusive, overwhelming evidence indicates that malfunction of α-syn, especially the aggregation of misfolded α-syn, plays an important role in the process of neurodegeneration [5, 8].

Neuronal expression of either human wild-type or PD-related mutant α-syn induces neurodegeneration associated with pathological accumulations of α-syn and reactive astrogliosis [9–Behavioral test

Rotarod test: as described previously [53], mice were placed onto a rotating rod with auto acceleration from 0 rpm to 40 rpm in 2 min (San Diego Instruments, San Diego, CA). The length of time the mouse stayed on the rotating rod was recorded.

Open-field test: as described previously [53], the ambulatory, fine and rearing activities of mice were measured by the Flex-Field activity system (San Diego Instruments, CA). Flex-Field software was used to trace and quantify mouse movement in the unit as the number of beam breaks per 30 min.

Grip strength measurement: as described previously [54], mice were allowed to use their forepaws or hind paws to pull or compress a triangular bar attached to a digital force gauge (Ametek, Largo, FL) set up to record the maximal pulling or compressing force. Five measurements were taken for each animal during each test.

Histology and Immunohistochemical Analyses

As described previously [54], mice were perfused via cardiac infusion with 4% paraformaldehyde in cold PBS. To obtain frozen sections, brain and spinal cord tissues were removed and submerged in 30% sucrose for 24 h and sectioned at 40 μm thickness with cryostat (Leica CM1950). For paraffin sections, sections at 8 μm thickness were obtained according to standard procedure. Antibodies specific to glial fibrillary acidic protein (GFAP) (1:1000, Sigma-Aldrich USA, St. Louis, MO), ionized calcium binding adaptor molecule-1 (Iba1, 1:1000, Wako Chemicals USA, Richmond, VA), tyrosine hydroxylase (TH, 1:1000, Pel-Freez Biologicals, Rogers, AR), α-synuclein (C20 &211, 1:1000, Santa Cruz Biotech, Santa Cruz, CA), Aquaporin 4 (1:500, Chemicon International, Inc USA, CA), glucose transporter 1 (1:500, Chemicon), von Willebrand Factor (1:500, Dako USA, Carpinteria, CA), iNOS (1:500, Sgima), SMI32 (Sternberger Monoclonal, Lutherville, MD), NeuN (1:1000, Chemicon) were used as suggested by manufacturers. Alexa 488 or Alex 568-conjugated secondary antibody (1:1000, Invitrogen) was used to visualize the staining, and Topro3 (1:1000, Invitrogen) was used for counterstaining the nuclei. Fluorescence images were captured using a laser scanning confocal microscope (LSM 510; Zeiss, Thornwood, NJ). The Images of 100 × objective (bar = 20 μM) were presented as a single optic layer after acquired in z-series stack scans at 0.8 μM intervals from individual field. The numbers of microglia and microglia cluster in the images taken from 25 × objective (368 μm × 368 μm) were counted.

Stereology

According to stereotaxic coordinates of mouse brains (3rd edition, Keith B.J. Franklin and George Paxinos), a series of coronal sections across the striatum (9 sections by every 10th section, Bregma -2.06-1.54 mm), SNpc (7 sections by every 4th section, Bregma -2.70- -3.82 mm), as well as cervical (approximate T1-T6) and lumbar spinal cord (approximate L1-L5, 10 sections by every 12th section) were stained with NeuN plus TH and NeuN, respectively, and visualized using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The number of NeuN or TH-positive cells was assessed using Stereo Investigator 8, an unbiased stereological procedure with an optical fractionator (MicroBrightField Inc, Williston, VT). The sampling scheme was designed to have coefficient of error (CE) less than 10% in order to get reliable results. All stereological analyses were performed under the 100 × objective of a Zeiss Axio microscope (Imager A1).

Primary cell cultures

Primary cortical neuron cultures were conducted as described previously [55] by using postnatal day 1 pups. For cortical astrocyte and microglia cultures [56], the dispersed cells were collected by centrifugation and plated on 75 cm2 flasks in DMEM supplemented with 10% fetal bovine serum (FBS). The cells were pre-incubated at 37°C in a humidified atmosphere of air/5% CO2 and the medium was changed first 24 h later and on alternate 3-days thereafter. After a pre-culture period of 8-11 days the cellular debris, microglia were lifted from astrocytes layer by shaking the culture flasks at 190 rpm for 3 h at 30°C. The cells attached to the flask were passed and grown in six-well plates for 3 days until harvest for astrocytes culture. The cells floating in the medium were collected by centrifuge and plated on 6-well plates in DMEM supplemented with 10% FBS for microglial culture. After 24 h incubation, the medium of microglial cultures was switched to conditioned medium from cultured astrocytes.

Tissue fractionation

As previously described (**an et al., in press), brain tissues (cerebral cortex, brainstem) were weighed and homogenized with 10 volumes of sucrose buffer (0.32 M sucrose, 1 mM NaHCO3, 1 mM MgCl2, and 0.5 mM CaCl2, plus protease and phosphatase inhibitor cocktails). Lysates were centrifuged at 1, 000 g for 10 min to separate supernatant (S1) and pellet (P1). Protein concentrations in S1 were measured by BCA (Pierce Biotechnology, Rockford, IL). S1 contains total α-synuclein protein, representing the sucrose fraction. An aliquot of S1 was diluted in the same volume of Triton extraction buffer (2% Triton X-100, 20 mM HEPES, plus protease and phosphatase inhibitor cocktails), homogenized by sonication, and centrifuged at 20, 000 × g for 30 min to obtain the Triton X-100-soluble (TX-sol) supernatant (S2) and Triton X-100-insoluble (TX-insol) pellet (P2). P2 was washed 4 times by 1% Triton X-100 buffer and centrifuged at 20, 000 × g for 10 min. The pellet fraction was further extracted in 2% SDS buffer (2% SDS, 20 mM HEPES, plus protease and phosphatase inhibitor cocktails) by sonication and centrifuged at 20, 800 × g for 5 min. The supernatant (S3) were present as Triton X-100-insoluble (TX-insol) or SDS-soluble fraction.

Western Blot

Proteins were size-fractioned by 4-12% NuPage BisTris-polyacrylamide gel electrophoresis (Invitrogen) using MOPS running buffer (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) or Nitrocellulose membranes. Antibodies specific to human/mouse α-synuclein (SynC20 recognizing the C terminal of both human and mouse α-synuclein, 1:1000, Santa Cruz; Syn-1 recognizing both human and mouse α-synuclein encoding amino acids 1-100, 1:1000, BD Biosciences, San Diego) [35] and β-actin (1:5000, Sigma) as loading control were used in this study. Horseradish peroxidase conjugated secondary antibodies were from Jackson ImmunoResearch. Signals were visualized by enhanced chemiluminescence development (Pierce, Rockford, IL) and quantified by imageJ software (NIH).

COX-1 inhibition

For COX-1 inhibitor treatment, mutant mice and control littermates at 2 months of age were administrated with SC-560 (30 mg/kg; Cayman Chemical, Ann Arbor, MI, USA) or vehicle (40% dimethyl sulfoxide in 0.1 M phosphate buffer, pH 7.4) through intraperitoneal (IP) injection once a day for 7 days [34].

Quantitative real-time PCR array

RNA was harvested using Qiagen RNeasy mini kit and converted into first-strand cDNA using RT2 First Strand Kit (SuperArray Bioscience Corporation). Quantitative real-time PCR was performed using an ABI Prism 7900HT Fast Detection System (Applied Biosystems).

Statistical Analysis

Statistical analysis was performed using the Graphpad Prism 5 (Graphpad Software Inc. La Jolla, CA). Data are presented as Means ± SEM. Statistical significances were determined by comparing means of different groups using t-test or ANOVA followed by Post Hoc Tukey HSD test, and presented as *p < 0.05, **p < 0.01, ***p < 0.001.