Journal: eneuro

Article Title: Synaptojanin1 Modifies Endolysosomal Parameters in Cultured Ventral Midbrain Neurons

doi: 10.1523/eneuro.0426-22.2023

Figure Lengend Snippet: Figure 2. Reduced LAMP1 expression and puncta number in Synj1-deficient MB neurons. A, B, Immunofluorescence analysis of LAMP1 in the soma of Synj11/1 and Synj11/ MB neurons, MAP2 is used as neuronal marker. A, Representative images. B, Quantification results, N = 82/88 (Synj11/1/Synj11/) neurons. Extended Data Figure 2-1 shows reduced LAMP2 and LAMP2A in the soma of Synj1-deficient MB neurons. C, Quantification of LAMP1-labeled puncta for Synj11/1 and Synj11/ MB neurons. y-axis represents the normalized puncta number per unit cytoplasmic area, N = 82/88 (Synj11/1/Synj11/) neurons. D, Cumulative frequency graph shows the LAMP1 fluorescence intensity distribution of the puncta in soma between Synj11/1 and Synj11/ MB neuron. N = 2091/2873 (Synj11/1/Synj11/) puncta. E, F, Analysis of LAMP1-GFP puncta in the axons of Synj11/1 and Synj11/ MB neurons. E, Representative images. F, Quantification results, N = 25/31 (Synj11/1/Synj11/) axons. The p values for B, C, and F are from Student’s t test. The p value for D is from two-sample Kolmogorov–Smirnov test. Scale bar in all images: 10 mm. Extended Data Figure 2-2 shows the comparison of LAMP1/LAMP2A expression between TH1 and TH MB neurons.

Article Snippet: The anti-MAP2 Polyclonal Chicken Antibody (188006, 1:1000 for IF) and the anti-MAP2 Polyclonal Guinea Pig Antibody (188004, 1:1000 for IF) were purchased from Synaptic Systems.

Techniques: Expressing, Immunofluorescence, Marker, Labeling, Fluorescence, Comparison

Journal: eneuro

Article Title: Synaptojanin1 Modifies Endolysosomal Parameters in Cultured Ventral Midbrain Neurons

doi: 10.1523/eneuro.0426-22.2023

Figure Lengend Snippet: Figure 3. Synj1 deficiency leads to endolysosomal hyperacidification and enhanced enzymatic activity. A–D, Endolysosomal acidity assay using a ratiometric fluorescent probe pHLy. A, C, representative images showing the mTFP1 signal and mCherry signal in the soma (A) and axons (C) of MB neurons transfected with pHLy. The brightness and contrast were adjusted to the same range for mTFP1 and mCherry channels. B, D, Distribution analysis of the ratio (mCherry/mTFP1) for the pHLy-labeled puncta between Synj11/1 and Synj11/ MB neurons at the soma (B) and at the axons (D). N = 1151/1108 (Synj11/1/Synj11/) puncta for soma analysis. N = 432/579 (Synj11/1/Synj11/) puncta for axon analysis. E, F, Cathepsin-B activity analysis using the Magic Red dye. E, Representative images showing the Magic Red signal from live cell imaging and the MAP2 signal from post hoc staining for Synj11/1 and Synj11/ MB neurons. F, Quantification of the intensity of Magic Red signal between Synj11/1 and Synj11/ MB neurons. N = 78/159 (Synj11/1/Synj11/) neurons. Extended Data Figure 3-1 shows that Synj1 deficiency does not alter the Cathepsin B expression in MB neurons. G, H, Analysis of endolysosomal protease activity using BSA-DQ. G, Representative im- ages. H, Quantification results, N = 90/111 (Synj11/1/Synj11/) neurons. The p values for B and D are from two-sample Kolmogorov–Smirnov test, and those for F and H are from Student’s t test. Scale bar in all images: 10 mm.

Article Snippet: The anti-MAP2 Polyclonal Chicken Antibody (188006, 1:1000 for IF) and the anti-MAP2 Polyclonal Guinea Pig Antibody (188004, 1:1000 for IF) were purchased from Synaptic Systems.

Techniques: Activity Assay, Transfection, Labeling, Live Cell Imaging, Staining, Expressing

Journal: bioRxiv

Article Title: GPCR endocytosis rewires neuronal gene expression and cellular architecture

doi: 10.1101/2025.08.26.672159

Figure Lengend Snippet: (A) Left : Schematic of the workflow for generation of human iPSC-derived cortical neurons. Right : Fixed cell fluorescence microscopy staining of iNeuron cultures (DIV21) with antibodies for the cortical marker, NGN2 (green), and neuronal marker, MAP2 (red). (B) Representative images of neurons treated with vehicle (no drug, ‘ND’) or 1 μM Isoproterenol (Iso) for 2 h, fixed and stained with antibody for MAP2 three days later (left) ; quantification of numbers of crossings by Sholl analysis (right) . Data are mean of n = 21–25 cells from 3 independent experiments. (C) Left: Representative images of neurons stained for vGLUT1 as presynaptic marker (green) and PSD95 as postsynaptic marker (red). Right: Quantification of synaptic pairs based on colocalization analysis of vGLUT1/PSD95 puncta per 35 μ m . Neurons were treated with vehicle (‘ND’) or 1 μM Isoproterenol (‘Iso’) for 24 h. Data are mean of n = 12 cells from 3 independent experiments. Error bars represent standard error of the mean. **** = p < 0.0001 by unpaired Student’s t test. Scale bars, 50 μm in (B) and 10 μm in (C) .

Article Snippet: Chicken anti-MAP2 monoclonal antibody (Phosphosolutions, Cat #1099-MAP2), rabbit anti-NGN2 polyclonal antibody (Invitrogen Cat #PA5–78556), guinea pig anti-VGLUT1 polyclonal antibody (Millipore, Cat #AB5905), rabbit anti-PSD95 polyclonal antibody (Abcam, Cat #AB18258) were used at 1:1,000 for immunofluorescent staining.

Techniques: Derivative Assay, Fluorescence, Microscopy, Staining, Marker

Journal: bioRxiv

Article Title: GPCR endocytosis rewires neuronal gene expression and cellular architecture

doi: 10.1101/2025.08.26.672159

Figure Lengend Snippet: (A-B) Detection of active β2AR/Gαs on endosomal vesicles. Left : Representative confocal images of neurons expressing Flag-tagged receptor (red) and GFP-tagged conformational biosensors for detection of active β2AR (Nb80, green) (A) and Gαs (Nb37, green) (B) . 1 μM Isoproterenol (Iso) was added at t=0. Right : Line scan analyses of colocalization between the β2AR and the biosensor. Yellow line: line scan analysis of gray value in each channel. Arrows indicate colocalization. (C) Endocytosis is required for cAMP accumulation. Neurons were pretreated with vehicle (DMSO) or 30 μM Dyngo-4a for 30 min, then stimulated with 1 μM Iso for 5 min, lysed and cAMP levels were measured using an ELISA assay. Data are mean of n = 3 independent experiments. (D-E) Analyses of dendritogenesis and synapse formation. Representative images are shown for each condition. Neurons were fixed and stained for MAP2 (D) or vGLUT1/PSD95 (E) . Neurons were pretreated with vehicle (DMSO) or 30 μM Dyngo-4a for 30 min, then stimulated with 1 μM Iso or 100 μM cAMP analog (8Br-cAMP). Quantification of number of crossings by Sholl analysis (D) and synaptic pairs (E) is shown. Data are mean of n = 18–25 cells in (D) , n = 9–12 (E) from 2–3 independent experiments. Error bars represent standard error of the mean. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05 by two-way ANOVA with Sidak correction in (C) , one-way ANOVA with Dunnett correction in (D-E) . ND = no drug; Veh = vehicle. Scale bars, 10 μm in (A), (B), (E) and 50 μm in (D) .

Article Snippet: Chicken anti-MAP2 monoclonal antibody (Phosphosolutions, Cat #1099-MAP2), rabbit anti-NGN2 polyclonal antibody (Invitrogen Cat #PA5–78556), guinea pig anti-VGLUT1 polyclonal antibody (Millipore, Cat #AB5905), rabbit anti-PSD95 polyclonal antibody (Abcam, Cat #AB18258) were used at 1:1,000 for immunofluorescent staining.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: GPCR endocytosis rewires neuronal gene expression and cellular architecture

doi: 10.1101/2025.08.26.672159

Figure Lengend Snippet: (A-B) PKA and mTOR are required for β2AR-induced neuronal morphogenesis. Representative images of dendritogenesis and synapse formation analyses are shown. Neurons were fixed and stained for MAP2 (A) or vGLUT1/PSD95 (B) . Neurons were pretreated with vehicle (DMSO), 10 μM H89 or 100 nM Torin for 30 min, then stimulated with 1 μM Iso. Quantification of number of crossings by Sholl analysis (A) and synaptic pairs (B) is shown. Data are mean of n = 13–17 cells in (A) and n = 10 cells in (B) from 2–3 independent experiments. (C-D) Endosomal β2AR signaling and PKA are required for mTOR activation. Neurons pretreated with vehicle (DMSO), 10 μM H89, 30 μM Dyngo-4a or 100 nM Torin for 30 min, then stimulated with 1 μM Iso for 30 min. Representative Western blots of 4E-BP1 phosphorylation (Thr37/46) are shown. (E) Model: Endosomal GPCR signaling elicits morphogenesis through transcriptional and translational changes that together boost the cell’s protein synthesis capacity to provide key structural effectors. Error bars represent standard error of the mean. **** = p < 0.0001 by one-way ANOVA with Dunnett correction. ND = no drug; Veh = vehicle. Scale bars, 50 μm in (A) and 10 μm in (B) .

Article Snippet: Chicken anti-MAP2 monoclonal antibody (Phosphosolutions, Cat #1099-MAP2), rabbit anti-NGN2 polyclonal antibody (Invitrogen Cat #PA5–78556), guinea pig anti-VGLUT1 polyclonal antibody (Millipore, Cat #AB5905), rabbit anti-PSD95 polyclonal antibody (Abcam, Cat #AB18258) were used at 1:1,000 for immunofluorescent staining.

Techniques: Staining, Activation Assay, Western Blot, Phospho-proteomics