Article Figures & Data
Figures
- Figure 1.
Characterization of isolated exosomes. Schematic representation of the methods used to characterize the exosome preparations. Left, Representative high-resolution transmission EM image of NSCexo and MNexo, isolated using the ultracentrifugation method (scale bar, 0.1 μm). Middle, Representative NTA profile for NSCexo and MNexo showing the size and concentration of particles. Right, Representative dot blot comparing exosome markers expression in NSCexo, MNexo, and total homogenate from mouse parietal cortex (mPC-H). NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; NTA, nano tracking analysis.
- Figure 2.
TauO-induced memory deficit is rescued by NSCexo. A, Schematic of the experimental design. Mice were injected (intracerebroventricularly) with either 1 × 109 exosomes (NSCexo or MNexo) or ACSF 24 h before injection (intracerebroventricularly) of 3 μl of 0.55 μM TauO or PBS. Four hours later, they were subjected to the NOR memory test. B, Discrimination index during the training phase when the mice are exposed to two identical objects. One-way ANOVA (F = 1.580) and Tukey’s multiple-comparison test. n = 7–10 mice/group. Data are mean ± SD. C, Box plot representation of the discrimination index calculated as the ratio between time spent with the novel object and the time spent with the familiar object during the 2 and 24 h memory recall tests. Two-way ANOVA of repeated-measure data with Dunnett multiple-comparison test versus training. *p < 0.05; **p < 0.01; ***p < 0.005. n = 7–10 mice/group. Data are min–max. NSCexo, neural stem cell exosomes; MNexo, mature neuronal exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; ACSF, artificial cerebrospinal fluid; SD, standard deviation.
- Figure 3.
Representative movement traces of mice during the NOR testing. Representative movement traces of mice intracerebroventricularly injected with either PBS, NSCexo, or MNexo prior treatment with TauO or ACSF. Left panels show data from the training phase of the test; middle panels show data from the 2 h memory recall test; right panels show data from the 24 h memory recall test. N, novel object; PBS, phosphate-buffered saline; NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; TauO, Tau oligomers; ACSF, artificial cerebrospinal fluid.
- Figure 4.
TauO-induced suppression of LTP expression in the hippocampus is abolished by intracerebroventricular injection of NSCexo. A, Schematic of the experimental design: Mice were administered intracerebroventricular injections of either NSCexo, MNexo, or PBS. After 24 h, they were killed. Brain sections from these mice were subsequently treated with or without TauO. Field recordings of LTP were then conducted in the SC region of the hippocampus. B,D, Field potential recording of LTP, compared with the percentage of the baseline, from brain slices pretreated with NSCexo but not with MNexo, in the presence or absence of TauO, revealed differences in the slope of EPSPs. C, For each experimental condition, the amplitude of fEPSP during the final 10 min (from the 50th to 60th minute post-HFS) was averaged. TauO significantly reduced LTP in the brain slice taken from mice injected with either PBS or MNexo. Conversely, this suppressive effect was not seen in slices from mice that received the NSCexo injection. PBS, n = 7; NSCexo, n = 7; and MNexo, n = 6 (1–4 slices per mouse). *p < 0.05. Two-way ANOVA followed by Tukey's multiple comparisons test. NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; LTP, long-term potentiation; fEPSP, functional excitatory postsynaptic potential.
- Figure 5.
Application of TauO reduces the slope, while application of MNexo affects the fiber volley. The input–output (IO) plotted as slope (mV/ms) on the Y-axis as a function of fiber volley (FV in mV) on the X-axis is provided prior to HFS (pre-, clear circles) and after (post-, filled circles) for (A) PBS treated, (B) NSCexo treated, (C) MNexo treated. The panel on the left is untreated, while the panel on the right is from TauO treated. Slopes for (D) PBS treated, (E) NSCexo treated, (F) MNexo treated and FV as a function of stimulation (μA) for (G) PBS treated, (H) NSCExo treated, (I) MNexo treated provide additional insight into the role of TauO and MNexo treatment on synaptic strength.
- Figure 6.
TauO-induced LTP suppression in the hippocampus is abolished by mimics of miRNAs enriched in NSCexo. A, Schematic of the experimental design. MiRNAs (scrambled or mimics of miR-322, miR17, and miR-485) or ACSF (vehicle) were injected intracerebroventricularly into adult mice 24 h before killing. B, SC field recording of LTP (indicated as the percentage of the baseline in the slope of fEPSPs) was performed on brain slices in the presence of TauO (50 nM) or ACSF. TauO abolished LTP in ACSF-injected mice and in scrambled-treated mice but not in miRNAs combo-treated mice. C, Average for each condition of the fEPSP amplitude for the final 10 min (time points 50–60 min post-HFS). TauO significantly reduced LTP in brain slices from mice injected with vehicle or with scrambled miRNA, but not in brain slices from mice treated with combo miRNAs mimics. N = 4 mice/group (3 slices per mouse). **p < 0.01; ****p < 0.0001; one-way ANOVA followed by Dunnett multiple-comparison test. ACSF, artificial cerebrospinal fluid; ICV, intracerebroventricular; LTP, long-term potentiation; fEPSPs, functional excitatory postsynaptic potentials; TauO, Tau oligomers.
- Figure 7.
NSCexo reduce Tau and pTau accumulation in the hippocampus. Representative images and quantitative analyses of triple immunofluorescence staining for NeuN (magenta) in combination with total Tau (red) and pTau (AT8-green) revealed a significant decrease in Tau and pTau levels after treatment with NSCexo in DG, CA1, and CA3. Scale bar, 20 μm. Statistical analyses were made using one-way analysis of variance, following Tukey's multiple-comparison test. Values are expressed as the mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NeuN, neuronal nuclei; NSCexo, neural stem cells exosomes; DG, dentate gyrus.
- Figure 8.
NSCexo reduce Tau and pTau accumulation in the cortex. Representative images and quantitative analyses of triple immunofluorescence staining for NeuN (magenta) in combination with total Tau (red) and pTau (AT8-green) revealed a significant decrease in Tau and pTau levels after treatment with NSCexo in both the parietal and frontal cortex. Scale bar, 20 μm. Statistical analyses were made using one-way analysis of variance, following Tukey's multiple-comparison test. Values are expressed as the mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NeuN, neuronal nuclei; NSCexo, neural stem cells exosomes; SD, standard deviation.
- Figure 9.
NSCexo reduce hippocampal synaptic vulnerability to TauO. Exosomes (NSCexo or MNexo) or PBS were injected intracerebroventricularly 24 h before sacrifice. Brain synaptosomes were prepared from the hippocampus and challenged with preformed 5 nM TauO for 1 h followed by treatment with PK or vehicle for 30 min. After washing, the amount of TauO bound to synaptosomes was quantified by ELISA. A, Schematic of the experimental design; (B) quantification of total Tau; (C) quantification of internalized Tau after PK treatment. n = 8 mice/group (average of two independent experiments). *p < 0.05; **p < 0.01 versus PBS unpaired T test. Data are median. NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; PK, proteinase K.
- Figure 10.
Effect of NSCexo on synaptic TauO binding and internalization. Exosomes (NSCexo or MNexo) or PBS were injected intracerebroventricularly 24 h before sacrifice. Synaptosomes were isolated from the hippocampus and challenged with preformed labeled TauO488 at increasing concentrations or PBS for 1 h. After washing, synaptosomes were treated with PK or vehicle for 30 min, washed and analyzed by flow cytometry. A, Schematic of the experimental design: B,C, The percentage of synaptosomes positive for TauO488 with and without PK treatment. D–E, MFI of TauO488-positive synaptosomes with and without PK. Comparisons were performed within each group between data in the presence or absence of PK. *p < 0.05 (multiple unpaired T test). Synaptosomes were pooled from eight individual mice per group. N = 3 flow cytometry runs. Data are mean ± SD. NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; PK, proteinase K; SD, standard deviation.
- Figure 11.
NSCexo reduce the internalization of TauO into synaptosomes. A, Schematic of the experimental design. B–F, Flow cytometric analysis of the percentage of TauO488-positive synaptosomes (after being challenged with increasing concentration of TauO) derived from NSCexo-, MNexo-, or PBS-treated mice before and after PK digestion. G–K, Flow cytometric analysis of the MFI of TauO488-positive synaptosomes derived from NSCexo-, MNexo-, or PBS-treated mice before and after PK digestion. *p < 0.05; two-way ANOVA followed by Tukey's multiple-comparison tests. Synaptosomes were pooled from eight individual mice per group. n = 3 flow cytometry runs. Data are mean ± SD. NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; PK, proteinase K.
- Figure 12.
NSCexo reduce TauO internalization into hippocampus neurons in vitro. Hippocampal neurons generated by differentiation of adult rat hippocampus NSC were treated with NSCexo, MNexo, or PBS for 24 h before being challenged with TauO (2.5 μM) for 1 h. A, Representative confocal images (60× with 2 zoom) of neurons pretreated with PBS, NSCexo, and MNexo (hTau in red, βIII-tubulin in green, and the nuclei in blue; scale bar, 20 µm). B, Representative confocal images (60× w/7.5 zoom) showing the presence of hTau inside the neurites (scale bar is 2 µm). C, Representative confocal images (60× with 7.5 zoom) showing the presence of hTau in the neuronal cell bodies (scale bar, 5 µm). D, Quantification of the number of hTau puncta inside neurons (5 images acquired from 8 independent experiments). *p < 0.05; **p < 0.01; one-way ANOVA (F = 6.701) followed by Tukey’s multiple-comparison test. E, Quantification of the number of neurites without hTau puncta (5 images acquired from 5 independent experiments). *p < 0.05; ****p < 0.0001; one-way ANOVA (F = 22.82) followed by Tukey's multiple-comparison test. F, Quantification of the number of hTau puncta on neurites (5 images acquired from 5 independent experiments). *p < 0.05; **p < 0.01; one-way ANOVA (F = 7.975) followed by Tukey's multiple-comparison test. Data are mean ± SEM. NSCexo, neural stem cells exosomes; MNexo, mature neurons exosomes; PBS, phosphate-buffered saline; TauO, Tau oligomers; SEM, standard error of the mean.
- Figure 13.
A, Hippocampal neurons generated by differentiation of adult rat hippocampus NSC were treated with PBS for 24 h before being incubated with PBS for 1 h. Neurons were fixed and processed for immunofluorescence analysis using anti-hTau antibody and anti-βIII–tubulin. Left, Representative confocal images (460× with 2 zoom) of neurons pretreated with PBS (hTau in red, βIII-tubulin in green, and the nuclei in blue; scale bar, 20 µm). Middle, Representative confocal images (60× with 7.5 zoom) showing the absence of hTau inside the neurites (scale bar, 2 µm). Right, Representative confocal images (60× with 7.5 zoom) showing the absence of hTau in the neuronal cell bodies (scale bar, 5 µm). NSC, neural stem cells; PBS, phosphate-buffered saline. B, Quantification of the number of DAPI+ cells across the experimental groups (5 images acquired from 8 independent experiments). One-way ANOVA (F = 1.035) followed by Tukey’s multiple-comparison test.
