Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods
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Materials and methods
A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4 -/- ) mice in a CD1 genetic background [ ] were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Immunohistochemistry
Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019-19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl.
Immunofluorescence
Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.
Brain homogenate preparation
Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm [ ]. To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature [ ]. Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein.
Western blotting
The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [ ]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit an...
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A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4 -/- ) mice in a CD1 genetic background [ ] were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019-19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl.
Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.
Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm [ ]. To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature [ ]. Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein.
The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [ ]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit anti-phosphorylated Tau Ser396/Ser404 (PHF-1, 1:1000, Abcam, ab184951) then followed by overnight incubation at 4 °C. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, and signals were detected by chemiluminescence...
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