hnRNP A2/B1 is a choroid plexus and CSF vesicle protein.

<p>A. Nanoparticle tracking analysis of serum free media from human CPE cultures and size frequency distributions. Media contained 559.1 (x10⁹) EVs per mL (N = 3). B. The average size of an EV was 160.3 nm (N = 3). C. Transmission electron micrographs of CPE EVs. Scale bar: 100 nm. D. Immunoblot of the exosome protein ALIX from vesicles isolated from CPEs cultured in the presence or absence of serum. E. The consensus sequence contains features of a recently described exosome miRNA motif recognized by hnRNPA2/B1. F. Multiple sequence alignment of vesicle proteins indicates conservation of miRNA sequence. G. Immunoblot of CPE derived media in the absence of serum or media alone containing serum for ALIX H. Immunoblot of hnRNPA2/B1 from CPE derived media in the absence of serum or media alone containing serum for. I. Immunoblot of hnRNPA2/B1 from CSF EVs demonstrating an age dependent decrease. J. Quantification of immunoblots for hnRNPA2/B1. *: p = 0.0138 Error Bars: SEM.</p

Next generation sequencing of CSF vesicle RNA content from patients less than 2 years old.

<p>A. Reads aligned to the human genome as a distribution across chromosomes. B. Rank sum distribution of mapped reads as a function of relative abundance (FPKM). C. Frequency distribution of reads in relation to read length. D. Alignment of a novel short RNA, CUFF .1222 to Hg19 chromosome 17 as visualized by integrated genomics viewer. The sequence of Hg19 is seen on the X axis. The chromosomal alignment is shown on the Y axis with red delineating the boundaries of the alignment.</p

Temporal dynamics of CSF vesicle miRNAs.

<p>A. miRNA microarray heat-map of CSF vesicle content from patients less than 2 years or greater than 70 years where red is higher expression and green is lower expression. B. Rank expression of miRNAs based on microarray probe intensity. Red is microarray data from patients less than 2 years and black is from patients greater than 70 years. C. Log(₂) signal of the most abundant miRNAs. D. Hierarchical clustering of miRNA microarrays at different ages. E. Venn diagram of high abundance or (F.) low abundance miRNAs at different ages.</p

CSF EVs undergo temporal declines.

<p>A. CSF from patients ranging from 1 month to 85 years was collected and subjected to nanoparticle tracking analysis. The average size of EVs was unchanged. B. The average number of EVs was highest in patients less than 2 years and decreased by 10–15 years and 70 years. C. Overlay of size distribution of vesicles from patients less than 2 years, 10-15 years, and greater than 70 years. D–F. Size distribution averages with SEM depicted. G–I. Size distribution averages with area under the curve depicted. *: p<.01 Error Bars: SEM.</p

Rodent eCSF nanovesicle purification and protein expression.

<p>(<b>A</b>) Flow chart of the experimental design after CSF labeling with a tracer dye (fast green) and eCSF collection. (<b>B</b>) Histogram of size distribution of eCSF nanoparticles (per ml) determined by nanoparticle tracking analysis (NanoSight). The nanoparticles were obtained from e14 CSF from three rat litters (N = 3). The mean nanoparticle diameter was 77 nm and was obtained by Gaussian curve fitting. (<b>C</b>) Electron micrographs of rat embryonic purified nanovesicles. Scale bar: 30 nm. (<b>D</b>) Immunoblots for rat exosomal marker proteins CD63 and HSP70, and additional proteins known to be in exosomes. PTEN and PKM2. (<b>E</b>) Quantification of phosphoenol pyruvate kinase enzymatic activity determined from rat nanovesicles. (<b>F</b>) Quantification of IGF pathway-related proteins in nanovesicles isolated from e15 rat CSF using the phospho (p)-pathscan assay. Error bars: SEM. Experiments were reproduced with nanovesicles isolated from three litters (N = 3).</p

Nanovesicles from eCSF increase IGF-mTORC1 activity in eNSCs <i>in vitro</i>.

<p>(<b>A</b>) Control and nanovesicle-treated eNSCs <i>in vitro</i> immunostained for phospho-(p) S6 (red), and nestin (green), and counterstained for the nuclear marker DAPI (blue). (<b>B</b>) Zoom of the image in the white square in (A). (<b>C</b>) Number of phospho-S6-positive cells relative to total with or without nanovesicle application (N = 3 and 4 cultures, 2–4 litters for nanovesicle extraction). Experiments were reproduced in the presence of vehicle (DMSO) or 100 nM rapamycin (N = 3 each). (<b>D</b>) Relative total cell number (N = 3 each). (<b>E</b>) Percentage of nestin-positive eNSCs (N = 3 each). (<b>F</b>) Percentage of Ki67- and nestin-positive eNSCs (N = 9 control and 3 with nanovesicle). *: p<0.05, **: p<0.01, ***: p<0.001 with Student's t test or one way ANOVA. Scale bars: 200 µm (A and B). Error bars: SEM.</p

microRNA analysis of rat eCSF nanovesicles.

<p>(<b>A</b>) Heat map of microRNA microarrays from eCSF purified nanovesicles. The lighter the color (yellow) indicates higher expression whereas the darker the color (dark purple) is an indication of absence of expression. Values range from (log₂) 0.64 (bottom) to (log₂) 15.4 (top). The top 24 enriched microRNAs are listed on the right. (<b>B</b>) Rank expression of microRNAs based on microarray expression levels. (<b>C</b>) Quantitative (q) RT-PCR of exosomal RNA using selective exosomal microRNA primers or lacking primers (Neg CTL: negative control). <u>Bottom</u>: Corresponding end-point RT-PCR. (<b>D</b>) Bioinformatic analysis of microRNA interacting pathways. N = 4 litters of rats.</p

Analysis of human eCSF nanovesicles.

<p>(<b>A</b>) Western blots for CD63, CD81, and HSP70 on purified human eCSF nanovesicles. (<b>B</b>) Pathscan analysis of human eCSF nanovesicles. All except CC3 are statistically above background at p<0.05. n.s., not significant. (<b>C</b>) Rank expression of human microRNAs based on microarray expression levels. (<b>D</b>) Differentially expressed microRNAs identified by Significant Analysis of Microarray (SAM) are indicated and represented in the heat map for human nanovesicles (left) and rat nanovesicles (right). Expression values range from low (bright green) to intermediate (black) to high (red). (<b>E</b>) Bioinformatic analysis of microRNA interacting pathways identified in eCSF purified human nanovesicles. Red microRNAs: 16-fold enriched (not shared with rat microRNAs); pink microRNA: 16-fold enriched and shared with rat; black microRNAs: 4-fold-enriched in humans and 16-fold enriched in rats. N = 4 humans. Error bars: SEM.</p