Journal: Science (New York, N.Y.)

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1126/science.abb4853

Figure Lengend Snippet: (A) The CPTX concept. While Cbln1 induces synapse formation by binding to postsynaptic GluD2 and presynaptic Nrx(+4), NP1 induces clustering of postsynaptic AMPARs without inducing presynaptic elements. CPTX is a chimeric ESP consisting of Cbln1 and NP1 structural elements. (B) Crystal structure of the pentraxin domain of human NP1 (NP1PTX). The N- and C-termini are annotated. Calcium ions (Ca2+) and water molecules are shown as green and red spheres, respectively. 2mFo-DFc electron density is contoured at 1.0 σ. The Ca2+ coordination shell, which includes a cacodylate buffer molecule (CAC), is indicated by black lines. (C) Diagrams of CPTX and related constructs. CC, coiled-coil domain; PTX, pentraxin domain; CRR, cysteine-rich region; gC1q, globular C1q domain; 3Cl, triple coil. The graph on the right shows molecular masses of monomeric NP1PTX, trimeric NP1PTX-3Cl and hexameric CPTX. dRI, differential refractive index. (D) Binding isotherms for the interactions between the GluA1–4 or GluD2 ATDs and immobilized CPTX, NP1PTX and Cbln1. The GluA4 ATD interacted strongest with CPTX (K D ~ 4.5 ± 0.5 μM) and NP1PTX (K D ~ 11.0 ± 0.9 μM), but not with Cbln1. In contrast, the ATD of GluD2 only interacted with Cbln1 (K D > 100 μM). (E) Single-cycle kinetic sensorgrams for the interaction between the ectodomain of Nrx1β(±4) and immobilized CPTX. CPTX directly bound to Nrx1β(+4) (K D ~ 4.90 ± 0.90 nM), but not to Nrx1β(–4).

Article Snippet: Antibodies The origin, dilution, company and catalog number are as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, 2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCo, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or Guinea pig, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Dr. P. Scheiffele, University of Basel, Basel, Switzerland), anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020), anti-MAP2 (Goat, 1:500, Frontier Science, Af860), anti-PSD95 (Mouse, 1:500, Invitrogen, MA1-046), anti-pan-AMPAR (Guinea pig, 1:500, Frontier Institute, Af580), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-GluN1 (Mouse, 1:500, BD Biosciences, 556308 or Mouse, 1:500, Chemicon, MAB363), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620 or Guinea pig, 1:500, Frontier Institute, Af1000), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003 or Mouse, 1:500, Enzo Life Sciences, SAP7F407).

Techniques: Binding Assay, Construct

Journal: Science (New York, N.Y.)

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1126/science.abb4853

Figure Lengend Snippet: (A) Schematic depiction of SCI caused by hemisection at the 10th thoracic vertebra (T10). The lower panels show representative horizontal sections of the spinal cord immunostained for VGluT2, HIS (CPTX) and GluA4 upon Mock (vehicle) or CPTX injection. Scale bar, 0.5 mm. (B) Representative orthogonal images obtained by Airyscan super-resolution microscopy indicating the localization of CPTX in proximity of VGluT2- and GluA4-immunopositive puncta. Scale bar, 0.5 μm. (C) Representative immunohistochemical staining images of coronal sections stained for VGluT2 (blue), GluA4 (green) and HIS (CPTX; magenta) from mock- or CPTX-treated spinal cords. Scale bar, 5 μm. (D) Quantification of the fraction of GluA4+/VGluT2+-double-positive puncta. The bars represent the mean ± SEM. *P < 0.05, n = 16 slices from 8 mice, Student’s t-test. (E–G) Time-course analyses of locomotion (Basso Mouse Scale (BMS) score) in SCI mice after injections. Mock (vehicle), Chondroitinase ABC (ChABC), Cbln1 or CTPX were injected into the spinal cord immediately (E) or 1 week after (F) hemisection, or immediately after contusion by impactors (G, 70 kdyn impact force). For the sham controls, the spinal cord was surgically exposed without imposing injury or injection. Mice that showed a BMS score of 1.5 at 1 week after hemisection were selected for F. **P < 0.01, *P < 0.05, n = 9 mice for each treatment, repeated two-way ANOVA with post-hoc Bonferroni-Dunn test (comparing the various treatments with Mock for each time point).

Article Snippet: Antibodies The origin, dilution, company and catalog number are as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, 2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCo, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or Guinea pig, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Dr. P. Scheiffele, University of Basel, Basel, Switzerland), anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020), anti-MAP2 (Goat, 1:500, Frontier Science, Af860), anti-PSD95 (Mouse, 1:500, Invitrogen, MA1-046), anti-pan-AMPAR (Guinea pig, 1:500, Frontier Institute, Af580), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-GluN1 (Mouse, 1:500, BD Biosciences, 556308 or Mouse, 1:500, Chemicon, MAB363), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620 or Guinea pig, 1:500, Frontier Institute, Af1000), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003 or Mouse, 1:500, Enzo Life Sciences, SAP7F407).

Techniques: Injection, Microscopy, Immunohistochemical staining, Staining

Journal: Science (New York, N.Y.)

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1126/science.abb4853

Figure Lengend Snippet: (A) CPTX induced the accumulation of presynaptic terminals of cerebellar granule cells onto co-cultured HEK293 cells displaying AMPAR ATDs. The intensities of the synaptophysin immunoreactivity (Syp; magenta) onto HEK293 cells (green) are quantified in the lower graph. Mock, vehicle (HEPES buffered saline (HBS)) controls. The bars represent the mean ± SEM. ***P < 0.001, **P < 0.01, n = 16–22 fields from 7 independent experiments, one-way ANOVA followed by Tukey’s test. Scale bar, 20 μm. (B) Beads coated with CPTX induced the formation of presynaptic boutons positive for endogenous synaptophysin (Syp) and neurexins (Nrxs), and postsynaptic boutons positive for GluA1–3, in co-cultured hippocampal neurons transfected with GFP. Arrowheads indicate beads immuno-positive for Syp (magenta, middle panel), Nrxs (cyan) or GluA1–3 (magenta, lower panel). Mock, beads coated with anti-HIS antibody. TL, transmitted light. Scale bar, 5 μm. The intensities of the Syp, Nrxs and GluA1–3 immunoreactivity onto beads are quantified in the upper graphs. The bars represent the mean ± SEM. ***P < 0.001, **P < 0.01, n = 21 fields from 5 experiments, one-way ANOVA followed by Tukey’s test. (C) CPTX localizes at excitatory synapses. Representative immunocytochemical staining images show Nrxs or VGluT1 (green), GluA1–4 (magenta) and HIS-tagged CPTX (cyan) in hippocampal neurons. Dendritic spines indicated by yellow arrowheads are magnified. Mean intensities of HIS signals in the Nrx-positive (Nrx+) or VGluT1-positive (VT1+) areas were measured and compared to those in the areas overlapping with GluA1–3 signals (GluA/Nrx+ or GluA/VT1+). The bars represent the mean ± SEM. ***P < 0.001, n = 24–26 fields from 2–3 independent experiments, Student’s t-test with Bonferroni correction. Scale bars, 2 μm (left panels), 1 μm (magnifications).

Article Snippet: Antibodies The origin, dilution, company and catalog number are as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, 2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCo, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or Guinea pig, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Dr. P. Scheiffele, University of Basel, Basel, Switzerland), anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020), anti-MAP2 (Goat, 1:500, Frontier Science, Af860), anti-PSD95 (Mouse, 1:500, Invitrogen, MA1-046), anti-pan-AMPAR (Guinea pig, 1:500, Frontier Institute, Af580), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-GluN1 (Mouse, 1:500, BD Biosciences, 556308 or Mouse, 1:500, Chemicon, MAB363), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620 or Guinea pig, 1:500, Frontier Institute, Af1000), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003 or Mouse, 1:500, Enzo Life Sciences, SAP7F407).

Techniques: Cell Culture, Transfection, Staining