Journal: bioRxiv

Article Title: In situ generation of Aβ 42 oligomers via secondary nucleation triggers neurite degeneration and synaptic dysfunction in human iPSC-derived glutamatergic neurons

doi: 10.1101/2024.08.30.610591

Figure Lengend Snippet: (A) Schematic representation of the experimental timeline. iCell glutamatergic human neurons were differentiated for 28 days in vitro (DIV). On DIV29 and DIV31, neurons were treated either with Aβ 42 monomer, fibrils or monomer with fibrils at 10% of the monomer concentration. 48 h after the second treatment, samples were processed. (B) Figure representing how co-treating neurons with monomer and fibrils may promote secondary nucleation processes that drive the generation of new oligomeric species responsible for cellular dysfunction. (C) Representative pictures of the levels of Amytracker-positive aggregates and synapsin-1-positive glutamatergic neurons treated twice with 500 nM of monomeric Aβ 42 or 500 nM Aβ 42 and 50 nM of pre-formed fibrils. Scale bar = 100 µm. (D,E) Total area of Amytracker-positive aggregates (D) and total area of neurites positive for the synapsin-1 (Syn1) maker (E) of iCell glutamatergic neurons treated twice, every 48 h, with either seeded or non-seeded Aβ42 at concentrations of 250 nM or 500 nM. Each data point corresponds to the total area of amyloid aggregates or Syn1-positive signal per analysed picture. A total of 48-50 images were analysed per condition. Data are represented as fold change with respect to cells treated with buffer. Statistical differences were calculated by applying a one-way ANOVA analysis, using the Tukey’s test to correct for multiple comparisons (***p-value < 0.001). (F-H) Comparison on the electrophysiological activity profile of iCell glutamatergic neurons treated once (T1) or twice (T2) with buffer, 500 nM of Aβ 42 monomer or 500 nM of monomer and 50 nM fibril, on a MEA plate. The number of active electrodes (F), number of network bursts (G) and synchrony index (H) are depicted (n = 9, mean±SEM). T0 represents the electrophysiological activity parameters from the corresponding MEA wells before they were treated with buffer, monomer or monomer + fibrils, respectively. Statistical differences were calculated by applying a one-way ANOVA analysis per treatment group, using the Dunnett’s test to correct for multiple comparisons (*p-value < 0.033, ***p-value < 0.001).

Article Snippet: The following primary and secondary antibodies were used for immunostaining experiments: Mouse anti-Aβ/APP antibody (1:500, SigmaAldrich, #MABN10); mouse anti-synapsin 1 (1:1000, Synaptic Systems, #106011), rabbit anti-β-III-catenin (1:1000, Abcam, #ab52623); Alexa Fluor 555 goat anti-Mouse IgG (H+L) (1:1000, Thermo Fisher, #A21424), Alexa Fluor 488 goat anti-Rabbit IgG (H+L) (1:1000, Thermo Fisher, # A11001).

Techniques: In Vitro, Concentration Assay, Comparison, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Illustration of the workflow used for the identification of the VGluT3-associated mouse IHC proteome. A , organs of Corti of immature (P8, before hearing onset) and mature (P23, after hearing onset) wild-type mice were explanted, homogenized, and fractionated by differential centrifugation. The organ of Corti is the auditory sensory epithelium and contains one row of IHCs and three rows of OHCs. VGluT3 is primarily expressed in trafficking organelles (vesicles and endolysosomal compartments) of IHCs. B , IHC VGluT3-positive organelles were immunoisolated using a VGluT3-specific antibody. C , subcellular fractions and immunoisolates were processed in a conventional bottom-up proteomics workflow. D and E , positive protein hits from the LC-MS/MS analyses were validated by immunohistochemistry using antibodies against VGluT3 and otoferlin to label IHCs, an antibody against RIBEYE/CtBP2 to label synaptic ribbons, and sometimes an antibody against synapsin-1 to label efferent synapses onto SGNs. D , representative mature IHCs at P15 immunolabeled for CtBP2 ( green ), VGluT3 ( red ), and otoferlin ( blue ) (scale bar: 5 μm); D1 shows higher magnification views of the basal region of one IHC (scale bar: 2 μm). The profile plot shows otoferlin’s immunofluorescence signal at the PM ( D2 ). E , representative P15 IHCs immunolabeled for CtBP2 ( green ), VGluT3 ( red ), and synapsin-1 ( blue ) (scale bar: 5 μm); E 1 shows higher magnification views of the basal region of one IHC (scale bar: 2 μm); the profile plot shows synapsin-1 signal beyond the IHC basolateral PM ( E2 ). In ( D and E ), maximum intensity projections of 5 to 10 confocal optical sections. In ( D 2 and E 2 ), fluorescence intensity line profiles through the longitudinal axis in the mid-region of a representative IHC, from apex to base (5–10 optical sections). ER, endoplasmic reticulum; IHC, inner hair cell; LC-MS/MS, mass spectrometry; OHC, outer hair cell; PM, plasma membrane; SV, synaptic vesicle; SGN, spiral ganglion neuron; VGluT3, vesicular glutamate transporter three.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Centrifugation, Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Immunolabeling, Immunofluorescence, Fluorescence, Mass Spectrometry, Membrane