Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Is Required for Normal Cochlear Function

doi: 10.1523/JNEUROSCI.0028-19.2019

Figure Lengend Snippet: BACE1 is expressed in spiral ganglion neurons and olivocochlear terminals. Expression of BACE1 was analyzed with immunohistochemistry on (A,B,E) cochlear cryosections and (C,D) whole-mount preparations of the apical cochlear turn of the organ of Corti. All images are representative maximum intensity projections of confocal z-stacks (see material and methods for details). A, We detected strong anti-BACE1 immunosignals in cell bodies of SGN in BACE1+/+ mice that (B) were strongly reduced in BACE1−/− mice. The panels (A+B) show representative transmitted light (left) and corresponding confocal images (right) of cryo-sections through the spiral ganglion stained with primary antibodies directed against BACE1 (green). C, In the organ of Corti of wild-type mice, we detected strong anti-BACE1 signals in synapsin1,2-positive structures that represent efferent olivocochlear terminals. D, These anti-BACE1 signals in efferent boutons were completely absent in the organ of Corti of BACE1−/− mice. C and D show confocal images of the organ of Corti in the IHC (top) or the OHC (bottom) region stained with primary antibodies against BACE1 (green) and synapsin1,2 (red). The right images in C and D show merged images of anti-BACE1 and the respective anti-synapsin1,2 staining. E, BACE1 expression was not detectable in the stria vascularis. We found comparable diffuse anti-BACE1-immunosignals in the stria vascularis of BACE1+/+ (left) and BACE1−/− (right) mice. We thus consider these signals as caused by unspecific binding of the primary antibody. F, As demonstrated with antibodies directed against KCNQ1 channels (KV7.1), localization and expression of KCNQ1 channels (green) in the stria vascularis was indistinguishable between BACE1+/+ (left) and BACE1−/− (right) mice. E and F show merged transmitted light and confocal images of cryo-sections through the stria vascularis. Scale bars, 20 μm.

Article Snippet: Immunostaining was performed overnight at 4°C with the following primary antibodies (diluted in blocking solution): rabbit-anti-BACE1 (Abcam, ab108394; 1:50), guinea pig-anti-synapsin1,2 (Synaptic Systems, 106004; 1:500), rabbit-anti-KCNQ1 (K v 7.1) (Abcam, ab135737; 1:200), goat-anti-prestin (N-20; Santa Cruz Biotechnology, sc-22692; 1:400), rabbit-anti-KCNQ4 (K v 7.4) (H-130; Santa Cruz Biotechnology, sc-50417; 1:400), rabbit-anti-NF-200 (neurofilament heavy chain polypeptide, Sigma-Aldrich, N4142; 1:600) and chicken-anti-NF-H (neurofilament heavy chain polypeptide, Abcam, ab4680; 1:400), mouse-anti-CtBP2 (C-terminal binding protein; BD Bioscience, 612044; 1:200), rabbit-anti-GluR2/3 (Glutamate receptor subunits 2/3, Merck, ab1506; 1:200), rabbit-anti-PSD95 (postsynaptic density 95, Abcam, ab18258; 1:200), mouse-anti-MBP (myelin basic protein; F-6; Santa Cruz Biotechnology, sc-271524; 1:400), and rabbit-anti-B-FABP (brain-type fatty acid binding protein, Kurtz et al., 1994 , 1:1000).

Techniques: Expressing, Immunohistochemistry, Staining, Binding Assay

Journal: eLife

Article Title: Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

doi: 10.7554/eLife.59323

Figure Lengend Snippet:

Article Snippet: Antibody , SYN1/2 guinea pig polyclonal antibody , Synaptic Systems , 106004 , Dilution (1:1000).

Techniques: Transfection, Generated, Purification, Marker, Recombinant, Plasmid Preparation, Control, Immunohistochemistry, Functional Assay, Software

Journal: Autophagy

Article Title: Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders

doi: 10.1080/15548627.2021.1936777

Figure Lengend Snippet: Synaptic phenotype in KdVS patient derived iNeurons. (A) Schematic presentation of the protocol to colocalize LC3 and SYN1-SYN2. Image of a dendrite of a control iNeuron at DIV21 after overnight incubation without B27 and treated with 200 nM BAF for 10 min before fixation. Scale bar: 10 µm. (B) Representative images showing dendrites stained for MAP2 and SYN1-SYN2 and SYN1-SYN2 puncta quantification at DIV21 for all lines. n = 60 for C 1 ; n = 57 for KdVS 1 ; n = 24 for CRISPR 1 ; n = 15 for C 2 ; n = 20 KdVS 2 ; n = 34 for KdVS 3 . Scale bar: 20 µm. One-way ANOVA and Sidak’s multiple comparison test were used to test for statistically significant differences. (C) Representative voltage clamp recordings at V h = −60 mV showing sEPSCs at DIV21. (D) sEPSC amplitude and (E) frequency quantification. n = 9 for C 1 ; n = 11 for KdVS 1 ; n = 10 for CRISPR 1 ; n = 15 for C 2 ; n = 8 for KdVS 2 and KdVS 3 (obtained in two independent experiments). (F) Schematic representation for neuronal network measurements on MEAs (3 min of recording). Representative raster plots for C 1 , KdVS 1 and CRISPR 1 derived networks that were plated at similar high densities, measured at DIV 30. (G) Quantification of the mean firing rate and (H) network burst rate, (I) percentage of random spikes, and (J) coefficient of variation (CV) calculated on the inter network burst interval. n = 15 for C 1 ; n = 18 for KdVS 1 ; n = 16 for CRISPR 1 . If not stated differently, data presented in this figure were obtained in at least 3 independent experiments and statistically significant differences were tested through Kruskal-Wallis and Dunn’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.

Article Snippet: Used primary antibodies were: rabbit anti-KANSL1 (1:500; Sigma-Aldrich, HPA006874); mouse anti-MAP2 (1:1000; Sigma-Aldrich, M4403); guinea pig anti-MAP2 (1:1000; Synaptic Systems, 188004); guinea pig anti-SYN1-SYN2 (1:1000; Synaptic Systems, 106004); rabbit anti-SYN1 (1:500; BioConnect, AB1543P); mouse anti-HOMER1 (1:500; Synaptic Systems, 160,011); rabbit anti-DLG4/PSD95 (1:50; Cell Signaling Technology, D27E11); mouse anti-pan axon (1:1000; Covance, SMI-312 R); rabbit anti-SQSTM1 (1:500; Sigma-Aldrich, P0067); mouse anti-LC3 (1:500; NanoTools, 0231–100/LC3-5 F10); rabbit anti-LAMP1 (1:200; Sigma-Aldrich, L1418-200ul); mouse anti-8-oxo-dG (1:100; R&D Systems, 4354-MC-050), rabbit anti-H4K16ac (1:400; Abcam, ab109463), rabbit anti-NANOG (1:100; Abcam, ab21624), mouse anti-SSEA4 (1:50; Abcam, ab16287), rabbit anti-POU5F1(1:250; Abcam, ab19857), mouse anti-TRA1-81 (1:100; Millipore, MAB4381) .

Techniques: Derivative Assay, Incubation, Staining, CRISPR

Journal: Autophagy

Article Title: Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders

doi: 10.1080/15548627.2021.1936777

Figure Lengend Snippet: Apocynin treatment rescues synaptic phenotype. (A) Representative images of 8-oxo-dG stainings and 8-oxo-dG quantification for untreated and APO treated iNeurons relative to respective untreated control cells at DIV21. n = 39 for C 1 ; n = 46 for C 1 + APO; n = 42 for KdVS 1 ; n = 51 for KdVS 1 + APO; n = 26 for CRISPR 1 ; n = 25 for CRISPR 1 + APO; n = 26 for C 2 ; n = 25 for C 2 + APO; n = 28 for KdVS 2 ; n = 27 for KdVS 2 + APO; n = 31 for KdVS 3 ; n = 23 for KdVS 3 + APO. Scale bar: 20 µm. (B) Representative images of SQSTM1 stainings of iNeurons at DIV21 and fluorescence quantification in untreated and APO treated iNeurons relative to untreated control cells at DIV21. n = 43 for C 1 ; n = 34 for C 1 + APO; n = 34 for KdVS 1 ; n = 38 for KdVS1+ APO; n = 40 for CRISPR 1 ; n = 39 for CRISPR 1 + APO; n = 16 for C 2 ; n = 19 for C 2 + APO; n = 25 for KdVS 2 ; n = 27 for KdVS 2 + APO; n = 34 for KdVS 3 ; n = 24 for KdVS 3 + APO. Scale bar: 20 µm. (C) Representative images of dendrites stained for MAP2 and SYN1-SYN2 for C 1 , KdVS 1 and CRISPR 1 at DIV21 either untreated or APO treated and SYN1-SYN2 quantification. n = 11 for C 1 ; n = 9 for C 1 + APO; n = 12 for KdVS 1 ; KdVS 1 + APO; n = 10 for CRISPR 1 ; CRISPR 1 + APO; n = 7 for C 2 ; C 2 + APO; KdVS 2 ; KdVS 2 + APO; KdVS 3 , KdVS 3 + APO. Scale bar: 20 µm. Two-way ANOVA was used to determine statistically significant changes. (D) Representative voltage clamp recordings at V h = −60 mV showing sEPSCs at DIV21 with and without APO treatment during differentiation. (E) sEPSC frequency and (F) amplitude quantifications. n = 8 for C 1 ; C 1 + APO; n = 9 for KdVS 1 ; KdVS1+ APO; n = 11 for CRISPR 1 ; n = 8 for CRISPR 1 + APO; n = 15 for C 2 ; n = 14 for C 2 + APO; n = 8 for KdVS 2 ; n = 9 for KdVS 2 + APO; n = 8 for KdVS 3 ; KdVS 3 + APO. (G) Representative raster plots for untreated C 1 network and untreated and APO treated CRISPR 1 network at DIV30 (3 min. of recording). Quantification of (H) mean firing rate, (I) network burst frequency, (J) percentage of random spikes, and (K) CV of inter-network burst interval. n = 15 for C 1 ; n = 17 for C 1 + APO; n = 16 for CRISPR 1 ; n = 15 for CRISPR 1 + APO. All data presented in this figure were generated in at least 2 independent experiments and statistically significant differences were tested through Kruskal-Wallis and Dunn’s multiple comparison test, if not mentioned differently. All samples were always compared to the respective untreated control; only significant differences were indicated. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.

Article Snippet: Used primary antibodies were: rabbit anti-KANSL1 (1:500; Sigma-Aldrich, HPA006874); mouse anti-MAP2 (1:1000; Sigma-Aldrich, M4403); guinea pig anti-MAP2 (1:1000; Synaptic Systems, 188004); guinea pig anti-SYN1-SYN2 (1:1000; Synaptic Systems, 106004); rabbit anti-SYN1 (1:500; BioConnect, AB1543P); mouse anti-HOMER1 (1:500; Synaptic Systems, 160,011); rabbit anti-DLG4/PSD95 (1:50; Cell Signaling Technology, D27E11); mouse anti-pan axon (1:1000; Covance, SMI-312 R); rabbit anti-SQSTM1 (1:500; Sigma-Aldrich, P0067); mouse anti-LC3 (1:500; NanoTools, 0231–100/LC3-5 F10); rabbit anti-LAMP1 (1:200; Sigma-Aldrich, L1418-200ul); mouse anti-8-oxo-dG (1:100; R&D Systems, 4354-MC-050), rabbit anti-H4K16ac (1:400; Abcam, ab109463), rabbit anti-NANOG (1:100; Abcam, ab21624), mouse anti-SSEA4 (1:50; Abcam, ab16287), rabbit anti-POU5F1(1:250; Abcam, ab19857), mouse anti-TRA1-81 (1:100; Millipore, MAB4381) .

Techniques: CRISPR, Fluorescence, Staining, Generated

Journal: iScience

Article Title: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

doi: 10.1016/j.isci.2022.104692

Figure Lengend Snippet:

Article Snippet: Guinea pig polyclonal antibody anti-synapsin1/2 , Synaptic Systems , Cat#106004; RRID: AB_1106784.

Techniques: Virus, Recombinant, cDNA Synthesis, Western Blot, Stripping, Immunodetection, Plasmid Preparation, Isolation, Sequencing, Software

Journal: bioRxiv

Article Title: Impaired neurogenesis and synaptogenesis in iPSC-derived Parkinson’s patient cortical neurons with D620N VPS35 mutation

doi: 10.1101/2024.08.07.606995

Figure Lengend Snippet: ( a , d , g ) Dendritic filament length (sum), number of branching points and average numbers of dendrites intersecting Sholl circles up to 200 μm distance per subgroup over time. ( b, e, h ) Group comparison between control and PD patient cell-lines. ( c ) Representative fluorescence images for human autaptic neurons for each subgroup at 4 WID. ci and cii are control neurons and ciii and civ are neurons induced from PD cell-lines. For immunohistochemical staining, MAP2 (green) was used as dendritic, Synapsin1/2 (red) as presynaptic and Shank2 (blue) as postsynaptic marker.

Article Snippet: The primary antibodies used were: MAP2 (chicken, 1:1000, Novus Biological), SHANK2 (guinea pig, 1:250, Synaptic Systems), Synapsin 1/2 (rabbit, 1:500, Synaptic Systems).

Techniques: Comparison, Control, Fluorescence, Immunohistochemical staining, Staining, Marker

Journal: bioRxiv

Article Title: Impaired neurogenesis and synaptogenesis in iPSC-derived Parkinson’s patient cortical neurons with D620N VPS35 mutation

doi: 10.1101/2024.08.07.606995

Figure Lengend Snippet: (a) Development of the number of synapses per subgroup over time. ( b ) Group comparison of controls vs PD patient cell-lines. ( c, d ) Representative fluorescence images for human autaptic neurons for each subgroup at 4 WID. Synapse number were counted by colocalizing Synapsin1/2 puncta (presynaptic marker, red) and Shank2 puncta (postsynaptic marker, blue) within proximity to dendrites (MAP2, green). Control cell ( c ) and PD patient cell ( d ) with magnification of synapse: i MAP2, Synapsin 1/2, Shank2, ii Synapsin 1/2, Shank2; iii Synapsin 1/2; iv Shank2.

Article Snippet: The primary antibodies used were: MAP2 (chicken, 1:1000, Novus Biological), SHANK2 (guinea pig, 1:250, Synaptic Systems), Synapsin 1/2 (rabbit, 1:500, Synaptic Systems).

Techniques: Comparison, Fluorescence, Marker, Control