Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Accumulation of aggregated α-syn in Lrrk2 astrocytes. A Schematic outline of the experimental setup. Cells were exposed to 0.5 μM sonicated Cy3 α-syn PFFs for 24 h. B TEM images of α-syn PFFs pre and post sonication. Scale bars 1 μm. C Representative fluorescence microscopy images of Lrrk1 +/+ , Lrrk2 −/− , and Lrrk2 GS/GS astrocytes (GFAP, green) at 24 h; cell nuclei stained with DAPI (blue) and α-syn labeled with Cy3 (red). Insets show a close-up of Cy3 α-syn inclusions. D Orthogonal projections of z-stack images taken with a fluorescence microscope: main view (x/y), top (x/z), and right (y/z). Projections were made along the lines depicted in the main image. Astrocytes (GFAP, green), Cy3 labeled α-syn (red), DAPI (blue). E Fluorescence microscopy image showing the differently sized Cy3 α-syn inclusions observed: small dot-like inclusions (arrow head) and larger, cottony deposits (arrow). E’ Displays of the particle count obtained from the ImageJ analysis when including either all Cy3 α-syn deposits or only the small Cy3 α-syn inclusions. Scale bars = 20 μm (C, D, and E)

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Sonication, Fluorescence, Microscopy, Staining, Labeling

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Analysis of the differently sized α-syn inclusions in Lrrk2 astrocytes. A , B Analysis of small and large Cy3 α-syn inclusions, respectively. Quantifications of Cy3 α-syn particle count, total area, and integrated density were performed using ImageJ. For both time points, ten images per independent cell culture (24 h: Lrrk1 +/+ , n = 7; Lrrk2 −/− , n = 6; and Lrrk2 GS/GS , n = 5; 24 h + 6 d: Lrrk1 +/+ , n = 6; Lrrk2 −/− , n = 6; and Lrrk2 GS/GS , n = 5) were analyzed and reported. For each time point, the statistical analysis was performed with the Kruskal-Wallis test followed by Dunn’s multiple comparisons test, since the data did not follow Gaussian distribution for all groups. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Cell Culture

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Internalization of aggregated α-syn in striatal astrocytes. A TEM images of SNARF-1 α-syn PFFs pre and post sonication. B Schematic outline of the experimental setup. Cells were exposed to 0.5 μM sonicated SNARF-1 α-syn PFFs for 24 h and imaged using live confocal laser scanning microscopy. C Representative images of primary Lrrk2 +/+ striatal astrocytes treated with SNARF-1 α-syn PFFs were acquired at range of 530–550 and 610–630 nm. Scale bar 50 μm. D , E Eight images per cell culture were analyzed ( n = 4 independent cultures). For each image, ROIs were traced and quantification of α-syn single-particle 550/630 ratio (IntDen) and number were performed using ImageJ. The cumulative distribution of the single-particle ratio was graphed for each genotype in D and particle number per ROI in E . Statistical analysis in E was performed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Sonication, Confocal Laser Scanning Microscopy, Cell Culture, Single Particle

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Description of the endo-lysosomal pathway in Lrrk2 striatal astrocytes. A Representative TEM image of primary striatal astrocyte section containing electron-dense lysosomal-like structures (arrows). Scale bar 2 μm. B , C Forty TEM images were acquired ( n = 4 per genotype). Each cell was imaged by covering the entire cytoplasm and lysosomal-like structure number and area were measured using ImageJ. D Representative image of the staining using Lamp2A (green) as a marker for the endo-lysosomal pathway, DAPI (blue) for the nuclei, and F-actin (cyano) to define cells. Scale bar 20 μm. Inset shows a close-up of Lamp2A-positive structures. Quantifications of Lamp2A-positive structure number and area were analyzed using ImageJ. E , F Four images per cell culture were analyzed ( n = 3 per genotype). G Measurement of lysosomal pH was done in primary striatal astrocytes from the three genotypes upon unlabeled α-syn PPF treatment ( n = 3 per genotype). Bafilomycin has been applied as negative control. Fluorescence ratio of light acquired at 535 nm upon excitation at 340 and 380 nm is provided. H Neutral red assay was performed in primary striatal astrocytes from the three genotypes upon unlabeled α-syn PPF treatment ( n = 4 per genotype). Bafilomycin has been applied as negative control. Absorbance at 540 nm measured upon cell lysates was normalized by total protein content. Statistical analysis in B , C , and F was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in E , G, and H was performed with one-way ANOVA followed by Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Staining, Marker, Cell Culture, Negative Control, Fluorescence, Neutral Red Assay

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Characterization of LRRK2 interactoma in stimulated condition. A Schematic outline of the experimental setup. H4 cells were transfected using 3xFlag-LRRK2 encoding plasmid and, after 48 h post transfection, treated with 0.5 μM sonicated unlabeled α-syn PFFs for 24 h. LRRK2 was subsequently immunopurified using anti-Flag agarose beads (IP), eluted with Flag peptide (elution), and subjected to LC-MS/MS analysis. FT, flow-through; unbound LRRK2. B Western blot analysis showing LRRK2 expression, immunopurification, and elution in H4 cells in treated and basal conditions. C Relative quantification of LRRK2 interactome under treated and untreated conditions. The area of the precursor ions identified by LC-MS/MS analysis was used as a quantitative measure of the protein content. The ratio between the area of the precursor ions of untreated and treated samples (normalized by the content of LRRK2) was then considered to highlight proteins showing a different affinity for LRRK2 in the two conditions ( n = 2). D H4 cells transfected with GFP-ANXA2 in α-syn PFF-treated condition verifying the proximity of internalized α-syn fibrils and transfected AnxA2. AnxA2-GFP (green), α-syn (red), DAPI (blue). Scale bar 30 μm

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Transfection, Plasmid Preparation, Sonication, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Immu-Puri, Quantitative Proteomics

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Investigation of AnxA2 function in astrocyte-mediated α-syn phagocytic clearance. A Unlabeled α-syn PFFs have been applied to primary astrocytes for 24 h. Projections verify the proximity of the internalized α-syn fibrils and endogenous AnxA2. Cell cytoskeleton (F-actin, cyano), α-syn (red), AnxA2 (green), DAPI (blue). Scale bar 20 μm. B Schematic outline of the experimental setup. Cells were transfected using 3xFlag-GFP-encoding plasmid together with scramble or AnxA2 siRNA. 48 h post transfection cells were treated with 0.5 μM sonicated α-syn PFFs for 24 h and imaged using confocal microscopy. C Representative images of primary striatal astrocytes transfected with scramble or Anxa2 siRNA together with a GFP-encoding plasmid in α-syn PFF-treated condition. Projections verify the proximity of the internalized α-syn fibrils; GFP (green), α-syn (red), DAPI (blue). Scale bar 30 μm. D Four images per cell culture were analyzed ( n = 3). Quantifications of α-syn PFFs fluorescent-positive puncta were performed using ImageJ (ComDet plug-in). E Western blot analysis of primary striatal astrocyte lysates transfected with scramble and AnxA2 siRNA under basal and PFF-treated conditions. Anti-Lrrk2, anti-α-syn, and anti-AnxA2 antibodies have been employed. F , G , H Quantification of band intensity was performed using ImageJ and normalized by β-actin ( n = 3). Statistical analysis in D was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in F , G , and H was performed with an unpaired t test or one-way ANOVA followed by Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Transfection, Plasmid Preparation, Sonication, Confocal Microscopy, Cell Culture, Western Blot

Journal: Molecular Neurobiology

Article Title: Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

doi: 10.1007/s12035-021-02327-8

Figure Lengend Snippet: Analysis of AnxA2 function in G2019S primary striatal astrocytes at endogenous level. A Western blot analysis of primary striatal astrocyte lysates under PFF-treated and basal conditions using anti-Lrrk2, anti-α-syn, and anti-AnxA2 antibodies. B , C , D Quantification of band intensity was performed using ImageJ and normalized by β-actin ( n = 4). E Representative images of Lrrk2 +/+ and Lrrk2 GS/GS astrocytes treated or not with α-syn PFFs and stained with anti-AnxA2 (green), anti-α-syn (red), F-actin (cyano), and cell nuclei with DAPI (blue). MLi-2-treated Lrrk2 GS/GS astrocytes are shown in the bottom panels. Scale bar 20 μm. Insets show a close-up of α-syn inclusions and re-localized AnxA2. F , G Eight images per cell culture were analyzed ( n = 3). Quantifications of Anxa2 puncta and AnxA2-α-syn PFFs proximity were performed using ImageJ. Statistical analysis in F and G was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in B , C , and D was performed with unpaired t test or one-way ANOVA followed by Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: To generate α-syn pre-formed fibrils (PFFs), in-house purified as in [ ] or commercial (Anaspec #AS-55555) human α-syn monomers reconstituted at a concentration of 5 mg/ml in sterile DPBS (Gibco) and sterile-filtered (Costar Spin-X centrifuge tube filters (Merck), 0.45 μm) were incubated on a shaker (IKA MS3 Basic or ThermoMixer F1.5 Eppendorf, 1000 rpm) at 37 °C for 7 days. α-syn PFFs were diluted to a stock concentration in sterile PBS and stored at −70 °C.

Techniques: Western Blot, Staining, Cell Culture