Fig 1: aS fibrils gradually destabilize membrane integrity resulting in neuronal dysfunction.a Representative confocal microscope images showing primary rat cortical neurons loaded with the calcein-AM probe for 10 min and then treated for 1 h with the indicated 0.3 µM aS species. Semi-quantitative analyses of the calcein-derived fluorescence signal in primary rat cortical neurons and SH-SY5Y cells. b Representative confocal microscope images showing the Ca2+-derived fluorescence in primary rat cortical neurons treated for 15 min with the indicated 0.3 µM aS species and then loaded with the Fluo-4 AM probe. Semi-quantitative analysis of the intracellular Ca2+-derived fluorescence in primary rat cortical neurons and SH-SY5Y cells. c Time-course analysis of the intracellular Ca2+-derived fluorescence in SH-SY5Y cells treated for the lengths of time indicated with OB*/SF/LF at 0.3 µM. d Representative confocal microscope images of human iPSC-derived dopaminergic neurons expressing MAP-2 (ab32454, Abcam) and TH (sc-25269, Santa Cruz Biotechnology) markers at 14–18 days of maturation (three independent experiments with one internal replicate). Approximately 75% of the cells are TH positive (estimated by immunostaining). Nuclei were stained with DAPI. e Representative confocal microscope images showing caspase-3-derived fluorescence in human iPSC-derived dopaminergic neurons treated for 24 h with the indicated aS species at 0.3 µM. f Semi-quantitative analysis of the caspase-3-derived fluorescence in human iPSC-derived dopaminergic neurons and SH-SY5Y cells treated for 24 h with the indicated aS species at 0.3 µM. f MTT reduction in primary rat cortical neurons and SH-SY5Y cells treated for 24 h with the indicated 0.3 µM aS species. In all panels data are expressed as the percentage of the value for untreated cells. Experimental errors are S.E.M. (n = 3 with two internal replicates and n = 4 with three internal replicates for cortical neurons and SH-SY5Y cells, respectively, in panels (a), (b); n = 4 in panel c with one internal replicate; n = 3 with two internal replicates and n = 4 with three internal replicates for iPSC-derived dopaminergic neurons and SH-SY5Y cells, respectively, in panel f; n = 6 with three internal replicates in panel (g)). Samples were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test relative to untreated cells (in panels a, b, f, and g, *P < 0.05, **P < 0.01, ***P < 0.001). A total of 200–250 cells (a–f) and 150,000–200,000 cells (g) were analyzed per condition.
Fig 2: Visualization of human cholinergic transmission with iAChSnFR.A Schematic of the differentiation of cholinergic neurons from human-induced pluripotent stem cells (hiPSCs). CAT anti-choline acetyltransferase staining (Abcam, #ab223346), MAP2 anti-microtubule-associated protein 2 staining (Abcam, #ab32454), DAPI 4′,6-diamidino-2-phenylindole nucleic acid staining (Sigma-Aldrich, D9542). Note the authentication of hiPSCs in the previous report [67]. B Imaging fluorescence responses of an iAChSnFR expressing human iPSC-derived neuron evoked by a train of 20 stimulating pulses delivered at 32 Hz. C Deconvolution microscopic analysis of three-dimensional spatiotemporal ΔF/F profiling of the iAChSnFR expressing human iPSC-derived neuron to local electrical stimuli. Note one isolated release site indicated by pink arrow in C. D Spatial profiling of the isolated release site indicated by the pink arrow in C with a pixel-wise maximal ΔF/F plot. Fitting the data points in this plot with a single exponential decay function (pink line) yields an estimated ACh spread length constant of 0.88 µm.
Fig 3: Immunohistochemistry of microislands and bulk (12 mm coverslip) culture. (A) Confocal images of immunolabelled hippocampal cultures grown in microislands (left panels) or on 12 mm diameter coverglass (right panels) at different developmental stages (days in vitro, or DIV) as indicated. Dendrites were labeled with MAP2 (white), presynaptic terminals with Synapsin-1 (red), excitatory postsynapses with PSD-95 (green), and nuclei using Hoescht 33342 (blue). (B) Zoomed in regions to show synaptic marker localization from images in (A) where areas are indicated by white boxes.
Fig 4: Chronic hypoxia promotes α-syn phosphorylation at ser129 and abnormal aggregation in the mouse hippocampus.A–F The levels of p-α-syn and α-syn in the RIPA-soluble and RIPA-insoluble fractions (8 M urea dissolved) from the hippocampi of mice in each group (Con, H1d, H3d, H7d, H14d) were detected by western blots with β-actin as the internal reference; Statistical analysis was performed. G The levels of p-α-syn and the neuronal marker Map2 in the hippocampus of the H14d group and Con group were detected by immunofluorescence. H The high-power three-dimensional imaging of p-α-syn immunofluorescence and the fluorescence intensity of each channel. Data are expressed as the mean ± SEM (one-way ANOVA). *P < 0.05, **P < 0.01, n = 3. Bar = 50 μm in G.
Fig 5: The effect of HA NGs@exosomes on neural synaptic plasticity. a Timeline and grouping information for experimental series investigating synaptic plasticity. b The dendritic spines density in Control, OVX-CUMS, and E-HA PACAP&E2 group was observed with Golgi-Cox staining. A small red triangle indicated spines. Scale bar: 10 μm. c Spine density in each group was quantified. Data were presented as mean ± SD (n = 3). d Immunofluorescence staining of PSD95, and MAP2 was shown in each group. Scale bar: 10 μm. e The relative intensity of PSD95 was quantified. f fEPSP-evoked synaptically in dendritic tuft by direct electrical stimulation was shown for 90 s. g Normalized fEPSP was quantified in each group. Data were presented as mean ± SD (n = 6). ##P < 0.01 vs. Control + E-HA NC; *P < 0.05, **P < 0.01 vs. OVX + CUMS + E-HA NC, (one-way ANOVA test)
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