Journal: Science Advances

Article Title: Monoallelic de novo AJAP1 loss-of-function variants disrupt trans-synaptic control of neurotransmitter release

doi: 10.1126/sciadv.adk5462

Figure Lengend Snippet: ( A ) AJAP1 immunofluorescence in midsagittal brain section. AJAP1 is abundant in hippocampus (Hc), cerebellum (Cb), cortex (Cx), striatum (St), and olfactory bulb (OB). Antibody AF7970 specificity was controlled using sections of Ajap1 −/− mice. ( B ) Confocal image of AJAP1 immunofluorescence in midsagittal hippocampus. CA1 and CA3 subfields, molecular layer (ML), granule cell layer (GCL) and the hilus of the dentate gyrus (Hil) are labeled. ( C ) RNA-FISH for AJAP1 and GRIA2 (GluA2), a marker of HMCs, on coronal sections of the dentate gyrus. Colocalization of AJAP1 and GRIA2 transcript fluorescence in the hilus (arrowheads) suggests AJAP1 expression by HMCs. ( D ) AJAP1 immunofluorescence labeling in the dentate gyrus. AJAP1 labeling is intense in neuropil of hilus (arrowheads) and on GluA2-positive cell bodies (arrows). ( E ) AJAP1, GluA2, and Bassoon immunofluorescence at HMCs. AJAP1 localizes to synaptic structures on mossy cell dendrites adjacent to the presynaptic marker Bassoon. ( F ) Subcellular distribution of AJAP1 immunofluorescence in cultured mouse hippocampal neurons at day in vitro 21 (DIV 21). AJAP1 displays a punctate distribution in dendrites (MAP2, arrowhead) and is absent from axons (Ankyrin-G, arrow). ( G ) Dendrites of cultured hippocampal neurons immunolabeled for AJAP1 and markers for glutamatergic (PSD-95, VGluT1) and GABAergic (Gephyrin, VGAT) synapses. Arrowheads indicate colocalization, arrows denote absence of colocalization. ( H ) Structured illumination microscopy (SIM) imaging of AJAP1 in DIV 21 cultured hippocampal neurons with colabeling of the presynaptic marker Bassoon and the postsynaptic markers Homer1 and Gephyrin. AJAP1 shows a punctate distribution in proximal dendrites and partly colocalizes with synaptic markers. In selected synapses (arrowheads), AJAP1 labeling is found between pre- and postsynaptic markers, consistent with localization at the postsynaptic membrane. Graphs represent perpendicular and parallel mean fluorescence intensity profiles along the three lines of selected synapses. Bar graphs in enlarged areas, 500 nm.

Article Snippet: The following primary antibodies were used: Sheep anti-AJAP1 (AF7970, R&D Systems), rabbit anti-GluA2 (AB1768-I, Merck), mouse anti-Bassoon (VAPS003, Stressgen), rabbit anti-Bassoon (141 003, Synaptic Systems), chicken anti-MAP2 (ab5392, Abcam), mouse anti–Ankyrin-G (75–147, Neuromab), mouse anti–PSD-95 (124 011, Synaptic Systems), rabbit anti-VGluT1 (135 303, Synaptic Systems), mouse anti-gephyrin (147 021, Synaptic Systems), rabbit anti-VGAT (131 003, Synaptic Systems), guinea pig anti-GB2 (322 205, Synaptic Systems), mouse anti-GB1a , rabbit anti-Synapsin1/2 (106 002, Synaptic Systems), mouse anti-β-TubulinIII (T8660, Sigma-Aldrich), mouse anti-FLAG (F1804, Sigma-Aldrich), mouse anti-Calbindin1 (300, Swant).

Techniques: Immunofluorescence, Labeling, Marker, Fluorescence, Expressing, Cell Culture, In Vitro, Immunolabeling, Microscopy, Imaging, Membrane

Journal: bioRxiv

Article Title: Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors

doi: 10.1101/2024.04.21.590488

Figure Lengend Snippet: (A-C) Strategy for directed differentiation of SOX6+/NG2+ progenitors into cortical output neurons (A), the NVOF multigene construct (B), and the experimental outline (C). (D) Representative images of control- and NVOF-transfected cells at 1-, 3-, 7-, and 16-days-post-transfection (DPT). Unlike control-transfected cells, NVOF-transfected cells lose progenitor morphology at 1 DPT, and progressively exhibit complex neuronal morphology, including a primary axon-like process and multiple dendrite–like processes. (E) Percentage of control-GFP and NVOF-transfected cells with neuronal morphology and TUJ1 expression (∼42% at 3 DPT and ∼74% at 7 DPT for NVOF, n=4, >200 cells/n). (F) Quantification of primary process length for NVOF-induced neurons at 3- and 7 DPT (n=3, >100 cells/n). (G) Representative morphology of NVOF-induced, TUJ1+ neurons at 7 DPT. Note the single axon, dendrite-like structures, and multiple axonal collaterals. (H) Representative images of NVOF-induced neurons at 16 DPT showing acquisition of elaborate dendritic morphology and highly intercalated axonal processes. (I) High-power representative images of individual NVOF-induced neurons at 16 DPT showing dendritic complexity and a single primary axon-like process for each neuron (red arrows). (J) Representative images of Neurog2 -induced neurons with multiple atypical axon-like processes. (GFP is pseudocolored for enhanced clarity of cell morphology.) (K) Representative images of Neurog2 -induced neurons expressing the axonal marker ANKYRIN-G (ANK3) by multiple neurites. (L) Quantification of neurons with single versus multiple axons in Neurog2 - and NVOF-induced neurons. At 7 DPT, 49% ±16% of Neurog2 -induced neurons have multiple, long axon-like processes, whereas a small number of such neurons exist after NVOF induction (9% ±5%) (n=5, >100 cell). See methods for details. (M-N) Representative images of NVOF-induced neurons at 7 DPT showing compartmentalized expression of the somato-dendritic marker MAP2, the somato-axonal marker Neurofilament-M, and the mature neuronal marker, NeuN. (O) Quantification of NVOF-induced, TUJ1+ neurons expressing MAP2 at 3 DPT (∼48%, n=3, >200 cells) and 7 DPT (∼93%, n=4, >200 cells), as well as NeuN at 7 DPT (66% ±16%, n=4, >100 cells). (Q) Volcano plot showing upregulation of neuronal genes in NVOF-induced neurons compared to control-transfected cells at 7 DPT (RNA-seq, n=3, biological replicates). (Q) Bar graph of RNA-seq data displaying upregulation of neuronal genes and downregulation of progenitor genes in NVOF-induced neurons at 7 DPT. Neurons exclusively upregulate glutamatergic genes, but not genes specific to alternate neuronal identities. Scale bars (D, G, H, J, M, N) 100 µm; (I) 50 µm. Error bars show standard deviations. ∗∗∗∗p < 0.0001, ***p < 0.001, **p < 0.01, t-test in (E, F, L). n.f. (no TUJ1+ cell found).

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-ANK3 (ANKYRIN-G), 1:250 (Santa Cruz, sc-12719); rat anti-BrdU, 1:500 (ACSC, OBT0030); rabbit anti-CSPG4 (NG2), 1:500 (Millipore, AB5320); rabbit anti-CTIP2, 1:500 (Abcam, ab28448); rat anti-CTIP2, 1:250 (Abcam, ab18465); rabbit anti-CUX1, 1:200 (Santa Cruz Biotechnology, sc-13024); rabbit anti-DARPP32, 1:250 (Cell Signaling Technology, 2306S); rabbit anti-FOG2, 1:250 (Santa Cruz Biotechnology, sc-10755); rabbit anti-FOXP2, 1:2000 (Abcam, AB16064); mouse anti-GABA, 1:200 (Sigma, A0310); mouse anti-GFAP, 1:1000 (Sigma, G3893); rabbit anti-GFAP, 1:1000 (Sigma, G9269); chicken anti-GFP, 1:1000 (Invitrogen, A10262); rabbit anti-GFP, 1:1000 (Invitrogen, A11122); mouse anti-HA, 1:1000 (Covance, MMS-101R); mouse anti-ISL1, 1:250 (Novus, H00003670); mouse anti-MAP2, 1:500 (Sigma, M1406); chicken anti-NESTIN, 1:2000 (Novus, NB100-1604); mouse anti-NeuN, 1:500 (Chemicon, MAB377); rabbit anti-NF-M, 1:200 (Millipore, AB1987); mouse anti-NEUROG2, 1:100 (R&D Systems; MAB3314); goat anti-OLIG2, 1:200 (R&D Systems, AF2418); rat anti-RFP, 1:500 (antibodies-online, ABIN334653); rabbit anti-PCP4, 1:500 (Proteintech, 14705-1-AP); rabbit anti-PDGFRB, 1:100 (Cell Signaling, 3169); mouse anti-PSA-NCAM, 1:200 (Chemicon, MAB5324); mouse anti-SATB2, 1:200 (Abcam, ab51502); rabbit anti-SATB2, 1:500 (Abcam, ab34735); rabbit anti-SOX6, 1:500 (Abcam, AB30455); goat anti-SOX10; 1:200 (Santa Cruz, sc-17342); rabbit anti-SYNAPSIN, 1:500 (Synaptic Systems, 106002); mouse anti-SYNAPTOPHYSIN, 1:500 (Millipore, MAB5258); rabbit anti-TH, 1:250 (Millipore, AB152); rabbit anti-TUBB3 (Tuj1), 1:1000 (Sigma, T2200); mouse anti-TUBB3 (Tuj1), 1:1000 (Biolegend, MMS-435P), rabbit anti-vGLUT1, 1:500 (Synaptic Systems, 135302); rabbit anti-2A-peptide, 1:1000 (Millipore, ABS31), rabbit anti-5HT, 1:3000 (Immunostar, 20080).

Techniques: Construct, Control, Transfection, Expressing, Marker, RNA Sequencing