Journal: medRxiv

Article Title: Alpha-synuclein misfolding as a fluid biomarker for Parkinson’s disease and synucleinopathies measured with the iRS platform

doi: 10.1101/2024.09.02.24312694

Figure Lengend Snippet: iRS-surface characterization by synthetic αSyn antigens. Panel A : The secondary structure sensitive Amide-I band absorbance of capture-antibody-bound αSyn-monomers (green, Stressmarq Bioscience Inc SPR-321), αSyn-oligomers (orange, Stressmarq Bioscience Inc SPR-466), and αSyn-PPFs (red, Stressmarq Bioscience Inc SPR-322) in PBS at high concentration of 500 ng/ml and scaled (x1.5-4) for comparison. Their structural differences are indicated by the significant wavenumber shift (cm -1 ) ranging from 1652 cm -1 for α-helical/random-coil (green) over 1647 cm -1 (orange) to β-sheet dominated structures absorbing at 1624 cm -1 (red). Panel B : The inertness measures on the blocking solution (BS) layer at high concentrations (500-5000 ng/ml) without the capture antibody. No signal is observed without the antibody on the blocking layer demonstrating sufficient inertness for pg-ng/ml concentrations of αSyn in CSF. Abbreviations: αSyn, alpha-synuclein; BS, blocking solution; PFF, pre-formed fibril.

Article Snippet: Briefly, αSyn stocks (Stressmarq, Cat. No. SPR-321), stored at -80 °C, were slowly thawed on ice.

Techniques: Concentration Assay, Comparison, Blocking Assay

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: SARS-CoV-2 differently affects SNCA mRNA expression and α-syn protein levels at 24 and 48 h post-infection in A549-hACE2 and CaLu-3 epithelial lung cells. A qPCR-based relative quantification of SNCA mRNA in Mock- and SARS-CoV-2-infected cells (MOI 0.05), either in the presence or absence of exogenous IFN-β, 24 and 48 h post-infection. Results are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed by applying Two-way ANOVA, followed by multiple testing correction. To avoid graphs overcrowding, p values are shown for statistically significant groups of interest only. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B and C Representative immunofluorescence images for SARS-CoV-2 Spike (S) and α-syn proteins in Mock- and SARS-CoV-2-infected (MOI 0.05) in A549-hACE2 ( B ) and CaLu-3 ( C ) epithelial lung cells 24 and 48 h post-infection, in the absence (CTR) or presence of IFN-β. Cells were fixed in 4% Formaldehyde solution for 15 min and permeabilized with 0.1% of Triton X-100 for 15 min. Images are representative of n = 3 independent experiments. Bars correspond to 20 μm

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Expressing, Infection, Immunofluorescence

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: A1 and A2 Exogenous administration of α-syn monomers increases SARS-CoV-2 replication in A549-hACE2 and CaLu-3 epithelial cells, which is prevented by IFN-β. Quantification of viral replication from cell supernatants through RT-qPCR for the SARS-CoV-2 N gene in infected A549-hACE2 ( A1 ) and CaLu-3 cells ( A2 ), 24 and 48 h post-infection (MOI 0.05). A1 In A549-hACE2 cells, exogenous α-syn monomers significantly increased viral replication compared with infected CTR both at 24 and 48 h post-infection. A2 In CaLu-3 cells the pro-viral effect of exogenous α-syn monomers was maximally detected at 24 h, while it declined at 48 h post-infection. In both cases, IFN-β, either alone or in combination with exogenous α-syn, completely prevented SARS-CoV-2 replication. Results show SARS-CoV-2 N gene copy numbers quantified from the RNA isolated from 100 μl of cell supernatants. Results are shown as mean ± SEM from n ≥ 5 independent experiments. Values for 24 and 48 h post-infection were analyzed by applying One-Way ANOVA followed by multiple testing correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B1 and B2 Representative immunofluorescence images of SARS-CoV-2 Nucleocapsid (N) and α-syn proteins in Mock- and SARS-CoV-2-Infected A549-hACE2 epithelial lung cells 48 h post-infection (MOI 0.05), in the absence and presence of IFN-β, exogenous α-syn monomers, or both. SARS-CoV-2 replication is enhanced in the presence of exogenous α-syn monomers, which impair accumulation of permeabilization-resistant α-syn species that are instead increased by IFN-β. B1 For visualization of large, permeabilization-resistant α-syn species, that potentially correspond to multimers or intracellular membrane-bound species, cells were fixed for 15 min in 4% Formaldehyde solution and permeabilized with 0.3% of Triton X-100 for 15 min. Bars correspond to 20 μm. B2 For better preservation, and visualization of small, highly-soluble α-syn species potentially corresponding to monomers, cells were fixed for 15 min in 4% Paraformaldehyde (PFA) and permeabilized with 0.1% of Triton X-100 for 10 min. Bars correspond to 20 μm. Images are representative of n = 3 independent experiments. C Quantification of SARS-CoV-2-N-positive cells in SARS-CoV-2-infected CTR vs α-syn-treated SARS-CoV-2-infected cells. Values are expressed as the percentage of N-positive cells ± SD calculated as the number of N-positive cells/total cells per microscopic field from n = 12 microscopic fields per experimental group, from n = 3 independent experiments. Data were analyzed by applying the Student’s t-test for comparison between VIRUS CTR and VIRUS α-syn groups. **p < 0.01

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Quantitative RT-PCR, Infection, Isolation, Immunofluorescence, Membrane, Preserving, Comparison, Virus

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: In HUVECS, absence of productive SARS-CoV-2 infection is associated with α-syn accumulation. A Quantification of SARS-CoV-2 replication in HUVECs in the absence and presence of α-syn and IFN-β (MOI 1). Results are shown as mean ± SEM from n = 4 independent experiments. Data were analyzed by applying Two-Way ANOVA. **p < 0.01, ***p < 0.001. B In the absence of productive viral replication, overall α-syn immunostaining in HUVECs is not decreased by either SARS-CoV-2 infection or the addition of exogenous α-syn monomers. Representative immunofluorescence images for SARS-CoV-2-Nucleocapsid (N), and α-syn in Mock- and SARS-CoV-2-Infected HUVECs 72 h post-infection (MOI 1), in the absence and presence of IFN-β, or exogenous α-syn monomers. Cells were fixed for 15 min in Formaldehyde solution, followed by 15 min permeabilization with 0.1% Triton X-100. Bars correspond to 20 μm. C In HUVECs, absence of productive SARS-CoV-2 infection is associated with the occurrence of juxtanuclear α-syn foci. 3D reconstruction of immunofluorescence images for SARS-CoV-2-Nucleocapsid (N), and α-syn proteins in Mock- and SARS-CoV-2-Infected HUVECs 72 h post-infection (MOI 1) shows the presence of juxtanuclear α-syn foci (arrows) which are induced by SARS-CoV-2 and potentiated by IFN-β and exogenous α-syn monomers

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Infection, Immunostaining, Immunofluorescence

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: SARS-CoV-2 synergizes with an excess of exogenously added α-syn monomers to reduce α-syn multimer:monomer ratio, which is prevented by IFN-β. A Representative western blot, and B quantification of total α-syn, α-syn monomers, α-syn multimers, and C α-syn multimer:monomer ratio in A549-hACE2 cells 48 h post- Mock and SARS-CoV-2 infection in the presence of exogenously added α-syn. Quantification was performed by normalizing to total protein (Loading control). Results show raw normalized values presented as mean ± SEM from n = 3 independent experiments. Multimer:monomer ratio is expressed as absolute value calculated as “multimers/monomers”. Data were analyzed by applying Two-Way ANOVA (for total, multimer, and monomer α-syn quantification) or One Way ANOVA (for multimer:monomer ratio). *p < 0.05, ***p < 0.001, ****p < 0.0001

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Western Blot, Infection

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: A Gene expression changes associated with the pro-viral and anti-viral effects of exogenous α-syn monomers and IFN-β, respectively . qPCR-based transcriptional analyses were performed in A549-hACE2 cells 48 h post-infection. Results are expressed as mean ± SEM from n = 3 independent experiments. Data for each gene were analyzed by One Way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SNCA synclein alpha; IFNB Interferon beta; STAT1 Signal transducer and activator of transcription 1; MX1 Myxovirus Resistance Protein 1; MX2 Myxovirus Resistance Protein 2; OAS1 2'-5'-oligoadenylate synthetase 1, RIG-I retinoic acid-inducible gene I; TNFA tumor necrosis factor alpha, TNFRSF1A tumor necrosis factor receptor superfamily 1A, TLR8 Toll-like receptor 8; Toll-like receptor 9 (TLR9). B SNCA mRNA expression is negatively correlated with viral titers at 3 and 5d post-infection of epithelial lung cells . The analyses were performed in CaLu-3 and A549-hACE2 cells at 3 and 5d post-infection with SARS-CoV-2 at 3 different MOI (0.01, 0.005, 0.001). Correlation between relative SNCA mRNA expression and SARS-CoV-2 N gene mRNA expression in A549-hACE2 and CaLu-3 cells was calculated through the Pearson’s r coefficient. Statistical significance was calculated through the two-tailed, paired Student’s t test . n = 12 for A549-hACE2 cells; n = 9 for CaLu3 cells. C1 and C2 SNCA mRNA expression is increased in human PBMCs stimulated with SARS-CoV-2 and is negatively correlated with SARS-CoV-2 replication in co-cultured CaLu-3 cells. C1 qPCR-based quantification of SNCA mRNA expression in in vitro SARS-CoV-2-exposed human PBMCs compared with Mock CTR. Statistical significance was calculated through the two-tailed, paired Student’s t test . The graph shows individual values (n = 8) with mean ± SD. ** p < 0.01. C2 Correlation analysis between relative SNCA mRNA expression within SARS-CoV-2-exposed PBMCs, and SARS-CoV-2 replication (N gene copy number) in co-cultured CaLu-3 epithelial cells. Correlation was calculated through the Pearson’s r coefficient. Statistical significance was calculated through the two-tailed, paired Student’s t test. n = 8

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Expressing, Infection, Two Tailed Test, Cell Culture, In Vitro

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: Summary cartoon depicting the opposite effects of extracellular α-syn monomers and IFN-β upon cellular internalization, and related α-syn multimer:monomer dynamics during SARS-CoV-2 infection of susceptible epithelial lung cells

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Infection

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