Journal: Cell Death & Disease

Article Title: TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons

doi: 10.1038/s41419-018-0369-4

Figure Lengend Snippet: a Representative recording of population spikes (each purple vertical line), and bursts (purple clusters with >4 spikes/s) from an individual electrode of a multichannel electrode array. Representative tracings show averages of spikes/second/electrode, and associated scatter plots show quantitation of spike and burst rates for ( b – e ) Control, ( f – i ) ADEV-IL-1β (particle dose of 50 ADEVs/cell), ( j – m ) ADEV-IL-1β+ Scrambled oligonucleotide (Scr In, 20 pmole), and ( n – q ) ADEV-IL-1β+ oligonucleotide inhibitors for miR-125 and miR-16 (Combined In, 20 pmole each). Data are mean ± SEM. Paired t -tests were performed to compare spike and burst rate of each electrode before and after treatment. *** p < 0.001 increased compared to baseline and ### p < 0.001 decreased compared to baseline

Article Snippet: In brief, hippocampal tissues were separately dissociated by gentle trituration in a calcium-free Hank’s balanced salt solution and centrifuged at 1000× g . Cells were resuspended in Neurobasal media (Gibco) containing B27 supplement (Thermo Fisher Scientific), 1% antibiotic/antimitotic solution (104 Unit of penicillin G/ml, 10 mg streptomycin/ml and 25 μg amphotericin B/ml) (Sigma), and plated at a density of 40,000 cells/ml in 96-well plates (Corning) coated with polyethyleneimine (Sigma) or 160,000 cells/ml in multichannel electrode array chambers (Harvard Apparatus) coated with polyethyleneimine (Sigma) and laminin (Sigma).

Techniques: Quantitation Assay

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Transmission electron microscopy (TEM) images of MVBs in WT and LRRK2 G2019S astrocytes. For illustration purposes, the red boxes in the top panel indicate MVBs in the cytoplasm of astrocytes and the lower panel shows a zoomed-in view of the MVBs. ( B, C ) Quantification of mean area ( B ) and size distribution ( C ) of MVBs identified in TEM images of WT and LRRK2 G2019S astrocytes. Data are sampled from at least 20 cells (≥40 MVBs) in each experimental condition; error bars represent mean + SEM for two independent biological samples ( B ). Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (****p<0.0001). ( D ) Representative confocal images of astrocytes labeled by immunofluorescence with the exosome marker CD63 (green) and the astrocyte marker CD44 (red). ( E ) Electron microscopy image showing immunogold labeling of CD63 (large gold) in astrocytes. Dashed lines delineate MVB membranes.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transmission Assay, Electron Microscopy, Two Tailed Test, Labeling, Immunofluorescence, Marker

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A, B ) Quantification of CD63 gold particles shown as the number of particles per µm 2 multivesicular body (MVB) ( A ) and relative frequency ( B ) in WT vs. LRRK2 G2019S astrocytes. Error bars represent mean + SEM. ( C ) Transmission electron microscopy (TEM) image of an astrocyte and astrocyte-secreted EVs. The dashed line indicates the astrocyte cell membrane, and the arrows indicate EVs. The red box and the zoomed-in view show EVs that appear to bud from the astrocyte membrane (white arrowheads). ( D ) Identification of exosome markers in EV-enriched fractions obtained from astrocyte conditioned media (ACM) using an exosome antibody array. Each circle on the membrane represents a preprinted antibody spot marker of exosome or cellular contaminant, and the table details the name of each antibody marker spotted on the membrane. ( E ) Cryo-electron microscopy (cryo-EM) images of astrocyte-derived EVs that display an unusual morphology. ( F ) Quantification of the number of CD63 + EVs secreted in WT and LRRK2 G2019S ACM by ELISA using ELISA standards calibrated by nanoparticle tracking analysis (NTA) as discussed in Materials and methods. Data are from at least three independent biological replicates; error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transmission Assay, Electron Microscopy, Membrane, Ab Array, Marker, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Representative images of WT and LRRK2 G2019S isogenic astrocytes labeled by immunofluorescence with CD63 (green), LRRK2 (purple), and DAPI nuclear stain (blue) ( Ai, Aiv ). The images were analyzed using Imaris software to identify CD63 + MVBs (green surfaces) colocalized with LRRK2 (purple dots) and show the localization of the nucleus (DAPI, blue) ( Aii, Av ). Zoomed-in images shows two populations of CD63 + surfaces: CD63 + /LRRK2 + (red arrowhead), and CD63 + /LRRK2 - (white arrowhead) ( Aiii, Avi ). ( B ) Percentage of CD63-labeled surfaces that are also LRKK2 positive in WT and LRRK2 G2019S astrocytes, quantified with Imaris software using object-based colocalization. Data are from three independent biological replicates, and ≥40 astrocytes (>3000 MVBs) were analyzed per experimental condition; error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant). ( C ) Immunogold electron microscopy shows the presence of LRRK2 (small gold, red arrowheads) inside and in the vicinity of CD63 + MVBs (large gold) in WT and LRRK2 G2019S astrocytes. The dashed lines indicate the contour of MVBs. ( D ) Distribution of CD63 + MVBs according to their number of internal LRRK2 gold particles. Data are sampled from at least 20 astrocytes (≥59 MVBs) in each experimental condition. The distribution is significantly different in LRRK2 G2019S astrocytes compared to WT astrocytes (p-value = 0.0084, chi-square test). ( E ) Quantification of the amount of LRRK2 in WT or LRRK2 G2019S EV-enriched fractions by ELISA. Data are from at least three independent biological replicates; error bars represent mean + SEM; statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Labeling, Immunofluorescence, Staining, Software, Two Tailed Test, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of WT and LRRK2 G2019S astrocytes labeled with the exosome marker CD63 (green), the astrocyte marker CD44 (red), and the nuclear marker DAPI (dark blue). The bottom images show the corresponding Imaris software rendering of CD63 + MVBs, color-coded by distance to the nucleus, from blue (closest) to white (farthest). The plain white lines indicate the cell boundary (outer line) and nucleus (inner circle). ( B–E ) Quantification of the distance of CD63 + MVBs from the nuclear membrane in WT and LRRK2 G2019S isogenic ( B, C ) or non-isogenic ( D, E ) astrocytes using Imaris software ‘vesicles distance to closest nucleus’ calculation. The violin plot shows the median (blue dashed line) and interquartile range (red solid line) ( B, D ). Data are from three independent biological replicates, 40–70 astrocytes (>3300 MVBs) were analyzed for each experimental condition. Statistical analysis was performed using a Mann–Whitney test (****p<0.0001).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Immunofluorescence, Labeling, Marker, Software, Membrane, MANN-WHITNEY

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) Immunogold labeling of phospho-S129 alpha-synuclein (p-αSyn, small gold, red arrowheads) and CD63 (large gold) shows localization of p-αSyn inside and in the vicinity of MVBs in astrocytes. The low abundance of small gold particles in the control sample is consistent with the observation that healthy brain tissues contain low levels of p-αSyn . The dashed lines indicate the boundary of the MVBs. ( B ) Distribution of CD63 + MVBs according to their number of internal p-αSyn gold particles, in WT and LRRK2 G2019S astrocytes. Data are sampled from at least 20 astrocytes (30–79 MVBs) for each experimental condition. The distribution is significantly different in LRRK2 G2019S astrocytes compared to WT astrocytes (p-value=0.0014, chi-square test). ( C ) Quantification of the amount of αSyn in WT and LRRK2 G2019S extracellular vesicle (EV)-enriched fractions by ELISA. Data are from seven independent biological replicates; error bars represent mean + SEM; statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. (ns: not significant).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Labeling, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: ( A ) WT dopaminergic neurons were transduced with rh10-CAG-tdTomato (red), and WT or LRRK2 G2019S astrocytes were transduced with lenti-CD63-GFP (green) to produce green-labeled exosomes. Neurons and astrocytes were co-cultured, and uptake of CD63-GFP exosomes by neurons was monitored by live-cell confocal microscopy, followed by deconvolution and Imaris modeling. ( B ) Confocal images of tdTomato neurons ( Bi ), CD63-GFP astrocytes ( Bii ), and the merged image ( Biii ). The corresponding Imaris software rendering represents tdTomato neurons in yellow ( Biv ), and the CD63-GFP exosomes in blue (outside the neurons) or purple (inside the neurons) ( Bv ). A transverse view of a neuron shows purple-labeled exosomes inside the somas and neurites ( Bvi ). ( C ) Quantification of the percentage of neurons with internalized WT or LRRK2 G2019S CD63-GFP exosomes at the time of live-cell imaging. ( D ) Quantification of the number of CD63-GFP exosomes per unit of neuronal volume. ( E ) Confocal images of tdTomato neurons co-cultured with WT or G2019S CD63-GFP isogenic astrocytes, and the corresponding Imaris software rendering representing neurons in white and CD63-GFP exosomes in red. White arrowheads show exosomes inside neurites. Scale bar: 5 µm. ( F ) Quantification of the number of CD63-GFP exosomes per unit of soma or neurite volume. For all datasets: data are from three independent biological replicates, ≥80 neurons were analyzed for each experimental condition. The scatter plot shows the median value. Statistical analysis was performed using two-tailed unpaired Student’s t-test with equal s.d. ( C, D ), or one-way ANOVA with Newman–Keuls multiple comparisons ( E ) (ns: not significant, *p≤0.05).

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Transduction, Labeling, Cell Culture, Confocal Microscopy, Software, Live Cell Imaging, Two Tailed Test

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet: Widefield image of co-cultured tdTomato neurons (red) and CD63-GFP astrocytes (green) ( i ). The dashed red box shows the neuronal soma, and the corresponding Imaris software rendering represents the tdTomato neuron in yellow ( ii, iii ), and the CD63-GFP EVs in blue (outside the neurons) or purple (inside the neurons). The white arrowhead shows the internal budding of the neuronal plasma membrane and endocytosis of extracellular CD63 + exosomes.

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Cell Culture, Software, Clinical Proteomics, Membrane

Journal: eLife

Article Title: The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease

doi: 10.7554/eLife.73062

Figure Lengend Snippet:

Article Snippet: Grids were incubated for 2 hr in the presence of anti-LRRK2 (1:20 dilution, Abcam, cat# ab133474) and anti-CD63 antibodies (1:40 dilution), or anti-p-αSyn antibody (1:20 dilution, Abcam, cat# ab51253) with mouse monoclonal anti-CD63 (1:40 dilution) in blocking buffer.

Techniques: Magnetic Beads, Control, Knock-In, Labeling, Library Quantification, DNA Library Preparation, cDNA Synthesis, Purification, SNP Genotyping Assay, Recombinant, Sequencing, Software