Journal: Nucleic Acids Research

Article Title: Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

doi: 10.1093/nar/gkab078

Figure Lengend Snippet: PS-ASO binding to RNase H1 can induce the interaction of RNase H1 with P54nrb. ( A ) Schematic presentation of the experimental procedures of co-isolation of P54nrb, PSF, or pre-complexed P54nrb/PSF with RNase H1. Full-length or domains of RNase H1 was bound to the beads, incubated with or without PS-ASO to allow binding of PS-ASO with RNase H1. After the removal of unbound PS-ASOs, P54nrb/PSF complex was incubated with beads and co-precipitated. ( B ) Western analyses of P54nrb and PSF co-isolated with MBP-H1 or GST-H1. The bait protein RNase H1 was also probed. ( C ) Western analysis of P54nrb co-isolated with different domains or full-length RNase H1, in the absence or presence of toxic (T, ASO 558807) or gap2 OMe modified safe (S, ASO 936053) PS-ASOs. Ponceau staining of bait proteins is shown in lower panels (C, D, E, F and H). ( D ) Western analysis of PSF co-isolated with different domains or full-length RNase H1, in the absence or presence of toxic (T) or safe (S) PS-ASOs. Note that the lower band of PSF is a truncated form of the protein. ( E ) Western analysis of P54nrb co-isolated with H1-delMit, in the absence or presence of toxic (T, ASO 558807) or safe (S, ASO 936053) PS-ASOs. ( F ) Western analysis of P54 co-isolated with H1-delMit upon binding of different toxic PS-ASOs and their non-toxic gap 2′-OMe modified PS-ASO counterparts, as in panel F. Two pairs of different ASOs were analyzed in the same experiment, but with different exposure times for the Western blot in the right panel. ( G ) The paired Tms of toxic and non-toxic gap 2′-OMe PS-ASOs in panel F determined in PTS. ( H ) Western analysis of P54nrb co-isolated with H1-delMit, in the absence or presence of different 3–10–3 cEt toxic (red) or safe PS-ASOs (listed in panel J). ( I ) The average level of coisolated P54nrb mediated by toxic versus safe PS-ASOs. ( J ) The Tms of toxic and safe PS-ASOs are determined in PTS. The average values and standard deviations from 4 duplicates are shown in related panels.

Article Snippet: Plasmids expressing GST-tagged RNase H1 full-length and different domains, P54nrb, and PSF were synthesized from GenScript and constructed on pGEX-6P1 vector.

Techniques: Binding Assay, Isolation, Incubation, Western Blot, Modification, Staining

Journal: Nucleic Acids Research

Article Title: Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

doi: 10.1093/nar/gkab078

Figure Lengend Snippet: PS-ASO induced interactions of RNase H1 with P54nrb or with PSF are affected by 2′-modifications of PS-ASOs. Co-precipitation was performed as in Figure , using GST-H1-NTD ( A and B ) or GST-H1-CTD ( C and D ) or GST-H1-delMit ( E and F ) as baits, in the absence or presence of different PS-ASOs of different 2′-modifications, or different ASO/RNA duplexes (ASOs are as in Figure , right panel). The beads were incubated with either P54nrb (A, C and E), or with PSF (B, D and F), and isolated proteins were detected by western analyses. Ponceau staining of the bait proteins is shown.

Article Snippet: Plasmids expressing GST-tagged RNase H1 full-length and different domains, P54nrb, and PSF were synthesized from GenScript and constructed on pGEX-6P1 vector.

Techniques: Incubation, Isolation, Western Blot, Staining

Journal: Nucleic Acids Research

Article Title: Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

doi: 10.1093/nar/gkab078

Figure Lengend Snippet: PS-ASO binding to P54nrb enhances the interaction between P54nrb and PSF, but does not induce the interaction between P54nrb and H1-CTD. ( A ) Schematic presentation of the experimental procedures of co-isolation of PSF with P54nrb. GST-P54nrb was bound to the beads, incubated without or with toxic (558807) or safe (936053) PS-ASO to allow binding of the PS-ASOs to P54nrb. After the removal of unbound PS-ASOs, PSF was incubated with beads and co-precipitated. ( B ) Western analysis of PSF protein co-isolated with P54nrb. P54nrb was probed. The signal intensity of PSF was quantified, normalized to P54nrb, and the ratios of PSF/P54nrb signals were compared with that in no-ASO control isolation and plotted. ( C ) Western analysis of proteins co-isolated from HeLa cell lysate through affinity selection using biotinylated PS-ASO 902459 then competitively eluted with PS-ASO or ASO/RNA duplex. P32 and Ku70 were probed as negative and positive controls, respectively. ( D ) Schematic presentation of potential modes of ASO-induced interaction between RNase H1 and P54nrb. ( E ) Western analysis of co-isolated P54nrb protein with H1-CTD. The H1-CTD was bound to beads, incubated with ss-PS-ASO (116847), followed by incubation of complementary RNA (XL279) or a control RNA (XL1052). After washing, the beads were incubated with P54nrb, and co-isolated P54nrb was analyzed by western analysis. Ponceau staining of the membrane was shown below the lanes. ( F ) Western analysis of H1-CTD co-isolated using GST-P54nrb as bait, similar to panel A. After PS-ASO binding to GST-P54nrb, H1-CTD was incubated with the beads, and co-isolated proteins were analyzed by western for RNase H1 or P54nrb. ( G ) Western analysis of H1-delMit co-isolated using GST-P54nrb as bait, similar to panel A. After PS-ASO binding to GST-P54nrb, H1-delMit was incubated with the beads, and co-isolated proteins were analyzed by western for RNase H1. Ponceau staining of the membrane was shown below the lanes.

Article Snippet: Plasmids expressing GST-tagged RNase H1 full-length and different domains, P54nrb, and PSF were synthesized from GenScript and constructed on pGEX-6P1 vector.

Techniques: Binding Assay, Isolation, Incubation, Western Blot, Control, Selection, Staining, Membrane

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Cholesterol sensing by TRKB (A) Identification of CARC motif (red) in the TM domain of TRKB, but not TRKA or TRKC. (B) Cholesterol promotes the effects of BDNF on TRKB autophosphorylation (TRKB:pY) at moderate, but inhibits BDNF at low or high concentrations (interaction: F[5,84] = 5.654, p = 0.0002; n = 6/group). Cultured cortical cells received cholesterol (15 min) followed by BDNF or cholesterol (15 min) and were submitted to ELISA for TRKB:pY. (C) β-cyclodextrin (bCDX, 2 mM, 30 min) prevents BDNF-induced increase in TRKB-PLC-γ1 interaction (TRK:PLCg1) (interaction: F[1,20] = 9.608, p = 0.0056, n = 6/group). (D) Pravastatin (1 μM, 3 days) also blocks the BDNF-induced increase in TRKB:PLC-γ1 interaction (interaction: F[1,19] = 11.23, p = 0.003; n = 5–6). ∗ p < 0.05 from the ctrl/ctrl group, #p < 0.05 from ctrl/chol0 group, data expressed as mean ± SEM of percentage from control group. (E) Microscale thermophoresis demonstrated direct interaction between GFP-tagged TRKB and cholesterol (15 min) in lysates from GFP-TRKB expressing HEK293T cells; mutation of Y433F blocks this interaction in MST (interaction: F[11,72] = 15.25, p < 0.0001, n = 4). (F) Fluoxetine-induced increase in TRKB surface exposure is blocked by bCDX (interaction: F[1,73] = 7.022, p = 0.0099, n = 19–20). (G–J) Structure of wild-type TRKB (G) in the absence of cholesterol and (H) at cholesterol concentrations of 20 mol% and (I) 40 mol%, and (J) for the heterodimer of TRKB.wt and TRKB.Y433F at 20 mol %. Related to systems 5–8 in and Figure S2 for distance and α values between C termini. See also , , and .

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Microscale Thermophoresis, Expressing, Mutagenesis

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Cholesterol sensing by TRKB, related to Figure 1 (A-E) MG87 cells were treated with β-cyclodextrin (bCDX), NGF, BDNF, or cholesterol and the levels of TRKB:PLC-γ1 or surface TRKB determined by ELISA. In MG87 cells expressing TRKB, (A) BDNF (10ng/ml/15min), but not NGF (50ng/ml/15min), increases the TRKB:PLC-γ1 interaction [treatment: F(2,21) = 46.24; p = 0.0001] measured by ELISA. The effect of cholesterol (20μM/15min) on (B) TRKB:PLC-γ1 coupling [interaction: F(1,20) = 59.49; p = 0.0001] and (C) surface positioning of TRKB [interaction: F(1,54) = 4.202; p = 0.04] is counteracted by pre-treatment with beta-cyclodextrin (bCDX; 2mM/30min). In MG87 cells expressing TRKA, (D) NGF, but not, BDNF, increases the TRKA:PLC-γ1 coupling [treatment: F(2,21) = 25.29; p = 0.0001]. (E) Lack of effect of cholesterol-induced TRKA:PLC-γ1 in cells expressing TRKA [interaction: F(1,20) = 0.25; p = 0.64]. (F) Rat cortical cells were treated with different concentrations of bCDX (30min), challenged by a combo of cholesterol+BDNF (15min), and the levels of TRKB:pY was determined by ELISA. β-cyclodextrin (mM/30min) reverses the block of BDNF-induced pTRKB (10ng/ml/15min) by high cholesterol concentration (100 μM/15min) [F(1,40) = 96.95, p < 0.0001, n = 6/group]. (G) Rat hippocampal cells were treated with pravastatin and BDNF, fixed and stained for actin. Effect of pravastatin (1μM/3days) on BDNF-induced neurite branching (10ng/ml/3days); interaction: F(1,13) = 4.967, p = 0.0441, n = 3-5]. (H-K) representative images of pravastatin effect on BDNF-induced branching. (L,M) Rat cortical cells were treated with pravastatin, cholesterol and BDNF, and the cell viability determined by CellTiterGlo. Pravastatin-induced cell death (2 μM/5days) is counteracted by co-incubation with cholesterol (20μM/5days) and BDNF (10ng/ml/5days) [interaction: F(1,164) = 10.895, p = 0.001, n = 20-24]. Data expressed as mean ± SEM of percentage from ctrl group. ∗ p < 0.05 from the control group (Fisher’s LSD). (N-Q) Rat cortical cells were treated with β-cyclodextrin (bCDX) or antidepressants, and the levels of TRKB:PLC-γ1 determined by ELISA. The pretreatment with bCDX (2mM/30min) prevents the increase in TRKB:PLC-γ1 (PLCg1) induced by (N) imipramine [interaction: F(1,20) = 14.71, p = 0.0010, n = 6/group], (O) ketamine [interaction: F(1,19) = 9.335, p = 0.0065, n = 5-6], (P) R,R-HNK [interaction: F(1,20) = 8.033, p = 0.0102, n = 6/group] or (Q) fluoxetine [interaction: F(1,20) = 8.035, p = 0.0103, n = 6/group]. ∗ p < 0.05 from the control group (Fisher’s LSD).

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Concentration Assay, Staining, Incubation, Control

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain, related to Figure 2 (A) Lysates from HEK293T cells transfected to express TRKB were submitted to ligand binding assay. BDNF interaction with TRKB is not altered by the Y433F mutation (n = 6/group). See schematics in S5A. (B,C) MG87 cells transfected to express TRKB were treated with BDNF and the levels of pTRKB determined by western-blotting. BDNF-induced phosphorylation of TRKB at (B) Y816 is prevented in the TRKB.Y433F mutant [interaction: F(1,47) = 6.688, p = 0.0129; n = 10-14], but the Y433F mutation does not affect BDNF-induced phosphorylation of TRKB at (C) Y515 residues in MG87 cells [interaction: F(1,33) = 0.1874, p = 0.6679; n = 9-10]. (D,E) N2A cells transfected to express luciferase-tagged TRKB and/or raft-restricted FYN, were treated with BDNF and submitted to PCA. (D) The BDNF-induced dimerization of TRKB is compromised by the Y433F mutation [interaction: F(1,42) = 11.08, p = 0.0018; n = 11-12]. (E) The BDNF-induced increase in TRKB interaction with FYN fragment in lipid raft is compromised by the Y433F mutation [interaction: F(1,44) = 20.96, p < 0.000; n = 12]. Data expressed as mean ± SEM of percentage from ctrl group. ∗ p < 0.05 from the control group (Fisher’s LSD). (F) N2A cells transfected to express TRKB were treated with BDNF and submitted to fractionation of membrane components. The Y433F mutation prevents BDNF-induced (10ng/ml/15min) translocation of TRKB to lipid-rafts in N2A cells (DRM: detergent-resistant membranes; 1 of 2 replicas). Rat cortical cells were treated with bCDX and fluoxetine, and the levels of surface TRKB determined by ELISA. Rat cortical cells were treated with fluoxetine or ketamine and submitted to immunoprecipitation of PLC-γ1 and western-blotting for TRKB and PLC-γ1. (G) Representative western-blotting of co-immunoprecipitation of PLC-γ1 and TRKB phosphorylated at Y816 in cultured cortical cells of rat embryo (1 of 2 replicas); lane 1: ctrl, 2: ctrl, 3: fluoxetine (10 μM/15min), 4: ketamine (10 μM/15min). (H) Rat cortical cells were preincubated with cholesterol (50uM) and fluoxetine, chlorpromazine, pimozide or flupenthixol (10uM) for 15min and challenged with BDNF (10ng/ml/15min). The levels of TRKB:pY were determined by ELISA [interaction: F(3,64) = 181.9, p < 0.0001, n = 9/group]. Data expressed as mean ± SEM of percentage from ctrl group. ∗ p < 0.05 from the control group (Fisher’s LSD). ∗ p < 0.05 from the control group (Fisher’s LSD).

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Transfection, Ligand Binding Assay, Mutagenesis, Western Blot, Phospho-proteomics, Luciferase, Control, Fractionation, Membrane, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Cell Culture

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Cholesterol sensing by TRKB, related to and (A) The distribution of the distance between the C-terminal residues of the monomers (center of mass L451-L453 Cα atoms (indicated with an arrow in Figure 1 ) are shown as violin plots. Increasing cholesterol concentration increases membrane thickness, which for the wild-type decreases the C-terminal distance. Y433F results in the disruption of the dimerization interface and the cross-like conformation. The parallel-like conformation of the WT-Y433F dimer appears to have a smaller hydrophobic length than that of the individual WT helices. Given at the bottom are average values for the membrane thickness (phosphate-phosphate distance) and the average angle between the helices ɑ. [Kruskal-Wallis: H = 27.8736; p < 0.001; n = 10/group]. (B) The effect of cholesterol concentration and the Y433F mutation on the stability of the interdimeric interface. The stability of the dimerization interface is characterized by a distribution of the distance between the monomers’ Cα carbons of G443 shown as violin plots for wild-type at different cholesterol concentrations and for the Y433F heterozygous mutant at 20 mol% cholesterol concentration (systems 1-4, ) [Kruskal-Wallis: H = 25.4385; p < 0.001; n = 10/group]. The results demonstrate that the Y433F mutation results in a total disruption of the A439-G443 dimerization interface. (C) The distribution of the distance between the C-terminal residues of the monomers (center of mass L439-L437 Cα atoms) in the TRKA transmembrane domain shown as violin plots. The results indicate that cholesterol concentration has no notable effect on the distance between the C-terminal residues of the two monomers in the TRKA TM dimer. In essence, TRKA is non-responsive to changes in cholesterol concentration (systems 12-14, ).

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Concentration Assay, Membrane, Disruption, Mutagenesis

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain (A) Fluoxetine (10 μM/15 min) and ketamine (10 μM/15 min) increased pTRKB.Y816 in cortical neurons immunoprecipitated with anti-PLC-γ1 (F[2,45] = 11.03, p = 0.0001, n = 16/group). (B) Fluoxetine facilitates BDNF-induced activation of TRKB under high cholesterol concentrations (interaction: F[2,132] = 5.15, p = 0.0070, n = 12/group) in cultured cortical cells. (C and D) Biotinylated fluoxetine binds to TRKB in lysates of TRKB expressing HEK cells (interaction: F[7,153] = 16.18, p < 0.0001; n = 6–14), but not (C) to TRKB.Y433F mutant or (D) to TRKB carrying the TMD of TRKA (TRKB/TRKA.TM) (interaction: F[7,80] = 43.75, p < 0.0001, n = 6/group). (E and F) Binding of biotinylated R,R-HNK (interaction: F[7,160] = 14.91, p < 0.0001; n = 6–14) (E) and tritiated imipramine (interaction: F[7,16] = 106.1, p < 0.0001; n = 2) (F) to TRKB, but not to TRKB.Y433F. Data expressed mean ± SEM of percentage of binding at 100 μM for fluoxetine and R,R-HNK or at 30 μM for imipramine. (G) Esketamine displaces the interaction of biotinylated fluoxetine (1 μM) with TRKB (n = 8/group). (H and I) Cholesterol facilitates the interaction of (H) biotinylated fluoxetine (F[5,30] = 7.198, p = 0.0002, n = 6/group)and (I) R,R-HNK (F[5,30] = 4.592, p = 0.0031, n = 6/group) with TRKB. (J) In situ PLA demonstrates close proximity between biotinylated fluoxetine and TRKB on TRKB-expressing N2A cells (red dots). (K) No PLA signal is seen in cells not expressing TRKB. Blue, DAPI; scale bar, 10 μm. (L) MST demonstrated direct interaction between fluoxetine and GFP-tagged TRKB (15 min) in lysates from GFP-TRKB expressing HEK293T cells (n = 4/group). Experimental traces depicted in the inset, vertical bars: blue, fluorescence cold; red, fluorescence hot. See also , , and .

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Immunoprecipitation, Activation Assay, Cell Culture, Expressing, Mutagenesis, Binding Assay, In Situ, Fluorescence

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Model of fluoxetine interaction with TRKB transmembrane domain The fluoxetine binding pocket at the dimeric interface of the TRKB transmembrane helices. (A) A representative snapshot showing fluoxetine in the crevice between the TRKB monomers. Fluoxetine is shown in licorice and the protein in cartoon representations. The side chains that interact with the drug are labeled and shown in licorice. (B) Fluoxetine binding involves lipid molecules, which provide a closed cavity for the drug. The protein is shown in green cartoon, the drug in van der Waals, and the lipids in licorice representations. (C) The chemical structure of fluoxetine. The atom names are labeled and the chemically equivalent atoms are indicated with an apostrophe. (D) The contact probability between drug heavy atoms and the interacting protein residues. The upper and lower panels correspond to the two different transmembrane helices (residues of the second helix are tagged with an apostrophe). Contact probabilities are calculated using a minimum distance cutoff of 5 Å (system 10). (E) The distributions of the distance between the center of mass L451–L453 Cα atoms of each monomer are shown for membranes with 20 mol % cholesterol (green; system 9), 40 mol % cholesterol with (blue; system 10) and without bound FLX (orange; system 7). See also and and .

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Binding Assay, Labeling

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain, related to Figure 2 Lysate from HEK293T cells expressing TRKB were submitted to ligand binding assays. (A) Schematic representation of the biotinylated fluoxetine interaction with immobilized TRKB. (B-J) Biotinylated fluoxetine (1 μM) interaction with TRKB is reduced by non-biotinylated (B) fluoxetine (n = 6/group), (C) imipramine (n = 8/group), (D) moclobemide (n = 10/group), (E) venlafaxine (n = 6/group), (F) ketamine (n = 8/group), (G) R,R-HNK (n = 8/group), but not reduced by (H) S,S-HNK (n = 8/group), (I) chlorpromazine (n = 8/group), isoproterenol (n = 8/group) or diphenhydramine (n = 8/group), or (J) BDNF (n = 6/group). (K) Biotinylated R,R-HNK (1 μM) interaction with TRKB is not reduced by S,S-HNK (n = 12/group). (L,M) Biotinylated fluoxetine interaction with (L) TRKA (n = 7/group) from MG87 cells, or (M) lysates from non-transfected HEK cells (n = 10/group) are negligible compared to TRKB. The interaction of biotinylated fluoxetine is not altered in (N) TRKB lacking most of the intra and extracellular domains (TRKB.T1ΔEC, n = 12/group), but it is reduced by (O) V437A and Y433F mutations, and partially attenuated by S440A (n = 6/group).

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Expressing, Ligand Binding Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB in the intact cells, related to Figure 2 (A) Schematic representation of the in situ proximity ligation assay (PLA) between TRKB and biotinylated fluoxetine. (B-G) HEK cells were transfected to express TRKB and farnesylated GFP and were exposed to biotinylated fluoxetine (10uM/15min). The cells were fixed in PFA and the PLA reaction was conducted in permeabilized cells. (B-D) No signal from TRKB-FLX interaction was observed when cells were not transfected to express TRKB. (E-G) positive signal of TRKB-FLX (PLA). Scale bar: 20 μm. Zoom in square: 2.5x.

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: In Situ, Proximity Ligation Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants and cholesterol promote membrane trafficking and TRKB-mediated plasticity, related to and (A) The fluorescence recovered after bleaching of GFP-TRKB in the neurite shaft of hippocampal neurons [n = 4-6; interaction: F(59,480) = 0.7580, p = 0.9061]. (B-E) Fluoxetine- and R,R-HNK-induced increase in the surface levels of GluR1 subunit of AMPA receptors are prevented by (B) ANA-12 [F(2,89) = 22.13, p < 0.0001, n = 15-16], (C) k252a [F(2,89) = 27.83, p < 0.0001, n = 15-16] in rat cortical cells, and by (D,E) the Y433F mutation of TRKB [fluoxetine: F(5,132) = 3.941, p = 0.0023, n = 12/group; R,R-HNK: F(5,132) = 5.022, p = 0.0003, n = 12/group] in mouse cortical cells. Data expressed as mean ± SEM of percentage from ctrl group. ∗ p < 0.05 from ctrl/TRKB.wt at the same dose. (F-I) Electrophysiological parameters of TRKB.Y433F mice. (F) TRKB.Y433F mutant mice display reduced theta-burst stimulus-induced changes in LTP [n = 6/group; interaction: F(61,610) = 5.466; p < 0.0001] but no changes in the (G) tetanic-stimulus-induced LTP [n = 5/group; interaction: F(60,480) = 0.1333, p > 0.9999], although a significant genotype effect was observed in (H) paired-pulse facilitation [n = 9/group; genotype: F(1,64) = 5.664, p = 0.0203; interaction: F(3,64) = 0.6356, p = 0.5948] and (I) input-output ratio [n = 9/group; genotype: F(1,96) = 6.388, p = 0.0131; interaction: F(5,96) = 0.3945, p = 0.8515] no interaction was identified. Data expressed as mean ± SEM of percentage from t0, baseline, or ctrl group. (J-K) Fluoxetine-induced (15mg/kg/7days in drinking water) increased performance in OLM was prevented in mice (J) heterozygous to BDNF [n = 4-7; interaction: F(1,18) = 6.878, p = 0.0173], but not in (K) animals lacking the serotonin transporter [n = 6/group; t(10) = 2.962, p = 0.0142]. ∗ p < 0.05 from ctrl.

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Membrane, Fluorescence, Mutagenesis

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants promote membrane trafficking of TRKB (A–D) Representative images of the spine and shaft fluorescence in (A) control, (B) BDNF-, (C) fluoxetine-, or (D) ketamine-treated rat hippocampal neurons (E18; DIV14) transfected with GFP-TRKB before (basal), immediately (bleached), and 2 min (recovery) after photobleaching (for analysis of neurite shaft recovery, see Figure S4 A). Scale bar, 1,000 nm. (E–J) Recovery of GFP-TRKB in dendritic spines is increased by (E and H) BDNF (20 ng/mL/15 min, TRKB.wt n = 17–27; interaction: F[62,2,604] = 5.435, p = 0.0001; TRKB.Y433F n = 27–39; interaction: F[52,3,328] = 0.4595, p = 0.99), (F and I) fluoxetine (1 μM/15 min, TRKB.wt n = 9–22; interaction: F[177,3,068] = 2.220, p = 0.0001; TRKB.Y433F n = 28–42; interaction: F[59,4,012] = 0.5555, p = 0.99), and (G and J) ketamine (10 μM/15 min, TRKB.wt n = 15–18; interaction: F[59,1,829] = 3.361, p < 0.0001; TRKB.Y433F n = 20–22; interaction: F[59,2,360] = 0.3995, p > 0.9999), but this is prevented in GFP-TRKB.Y433F expressing neurons; data expressed as mean ± SEM of percentage from t = 0. (K–N) Representative images of the BDNF-induced clusters of GFP-TRKB on the surface of MG87.TRKB cells. Scale bar, 250 nm. (O and P) BDNF (10 ng/mL/15 min) and fluoxetine (10 μM/15 min, TRKB.wt n = 365–593; TRKB.Y433F n = 232–547; interaction: F[2,2,717] = 4.305, p = 0.0136) (O) and cholesterol (20 μM/15 min) and ketamine (10 μM/15 min, TRKB.wt n = 282–7,413; TRKB.Y433F n = 258–765; interaction: F[2,2,731] = 11.15, p < 0.0001) (P) enhance the formation of clusters of GFP-TRKB on the surface of MG87.TRKB cells but not in the GFP-TRKB.Y433F-expressing cells. ∗ p < 0.05 from respective control (vehicle-treated) groups; #p < 0.05 from BDNF- or fluoxetine-treated wild-type group (Fisher’s LSD), clusters from 10 cells/group, and 10 regions of interest (ROI) per image, mean ± SEM of cluster area (nm 2 ). See also Figure S6 A.

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Membrane, Fluorescence, Control, Transfection, Expressing

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Binding to TRKB mediates the plasticity-related effects of antidepressants (A) Treatment with pravastatin (10 mg/kg/day in the drinking water for 14 days) attenuated the BDNF-induced LTP in the hippocampus of anesthetized rats (F[85,1,290] = 1.484, p = 0.0036, n = 8–9). (B) Fluoxetine promotes hippocampal neurogenesis in wild-type, but not in TRKB.Y433F mice (n = 7–9; interaction: F[1,30] = 4.691, p = 0.0384). Mice received bromodeoxyuridine (BrdU) injections at day 1, the BrdU incorporation was measured after 3 weeks of fluoxetine treatment (15 mg/kg/day for 21 days in the drinking water, orally [p.o.]). (C) Fluoxetine (10 mg/kg/day for 28 days, p.o.; n = 6), R,R-HNK (10 mg/kg i.p. injection every second day for 8 days, n = 4), and ketamine (10 mg/kg i.p. injection every second day for 8 days, n = 5) permitted a shift in ocular dominance in adult mice during 7 days of monocular deprivation (paired t test: fluoxetine: t[5] = 2.985, p = 0.0306; R,R-HNK: t[3] = 6.875, p = 0.0063; ketamine: t[4] = 6.517, p = 0.0029). *p < 0.05 between intrinsic signal imaging (IOS) sessions. (D and E) Fluoxetine (D) and R,R-HNK (E) fail to permit a shift in ocular dominance in TRKB.Y433F mice (fluoxetine: F[1,19] = 256.9, p < 0.0001, n = 9–12; R,R-HNK: F[1,20] = 12.47, p = 0.0021, n = 6/group). (F) Treatment with fluoxetine induced a shift in ocular dominance in response to 7 days of monocular deprivation, but this effect is prevented by pravastatin (interaction: F[1,10] = 5.221, p = 0.0454, n = 5–6). (G) R,R-HNK induced a shift in ocular dominance in response to 7 days of monocular deprivation, but this effect is prevented by pravastatin (treatment: F[1,9] = 9.044; p = 0.0148, n = 4–7). ∗ p < 0.05 from the control group in the same session, Fisher’s LSD. Data expressed as mean ± SEM. The black groups in plots (F) and (G) are also depicted in (C). See also and Figure S6 .

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Binding Assay, BrdU Incorporation Assay, Injection, Imaging, Control

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Binding to TRKB mediates the behavioral effects of antidepressants (A) Fluoxetine improves object location memory (OLM) in wild-type mice, but this effect was absent in the TRKB.Y433F mice (interaction: F[1,18] = 6.878, p = 0.017; n = 8–9). (B) Fluoxetine improved object location memory in wild-type mice, but this effect was prevented by pravastatin (interaction: F[1,14] = 6.504, p = 0.023, n = 4-5). (C) R,R-HNK improved object location memory in wild-type mice, but this effect was prevented by pravastatin (interaction: F[1,20] = 10.59, p = 0.0040, n = 6/group). (D and E) Fluoxetine (D) (treatment: F[1,23] = 5.433, p = 0.0289, n = 6–8) and ketamine (E) (treatment: F[1,23] = 24.26, p < 0.0001, n = 5–9) reduce immobility in the forced swimming test in TRKB.wt mice, but are ineffective in TRKB.Y433F mutants. (F) Fluoxetine facilitated the extinction of contextual conditioned fear, and this response is blocked by pravastatin (interaction: F[6,40] = 5.099, p = 0.0006, n = 6/group). (G and H) Fluoxetine (G) and ketamine (H) facilitate the extinction of contextual conditioned fear in the 8-min session, and this response is blocked in mice carrying the TRKB.Y433F mutation (fluoxetine: F[6,34] = 3.241, p = 0.0126; n = 5–6; ketamine: F[6,40] = 4.896, p = 0.0008; n = 5–7). ∗ p < 0.05 from the control group in the same session, Fisher’s LSD. Data expressed as mean ± SEM. See also Figure S6 .

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Binding Assay, Mutagenesis, Control

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet:

Article Snippet: Plasmid to express GFP-tagged TRKB where TM domain is substuted by TRKA.TM domain , Genscript , GFP-TRKB/TRKA.TM.

Techniques: Recombinant, In Situ, Stable Transfection, Expressing, Plasmid Preparation, Software