Journal: Molecular Psychiatry

Article Title: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior

doi: 10.1038/s41380-018-0260-9

Figure Lengend Snippet: Lack of SERT increases the number of functional PFC-to-DRN synapses. a rAAV-CAG-hChR2(H134R)-mCherry was bilaterally injected into the PFC of P4–P5 control or SERT-KO mice. Photograph showing mCherry expression after the PFC AAV injection (upper left). Optogenetic stimulation and electrophysiological patch clamp recordings were made starting at P28 in coronal sections containing the DRN, as shown by the photograph of the immunolabeling of PFC mCherry+ axons innervating to DRN 5-HT neurons, identified by the presence of the enzyme TPH2 (upper right). b Amplitude of optogenetically evoked EPSCs (oEPSCs) at synapses from PFC terminals onto DRN putative 5-HT neurons (left) and non-5-HT neurons (right) at various light stimulation intensities. In control (SERT Cre/+ ) (5-HT: n = 10 cells/5 animals; non-5-HT: n = 7 cells/4 animals); in SERT-KO (SERT Cre/Cre ) (5-HT: n = 10 cells/3 animals; non-5-HT: n = 6 cells/3 animals). Top: example traces at 9.8 mW (black/gray) and at 2 mW (red) stimulation); Bottom: input/output curves. Two-way ANOVA on 9.8 mW intensity: genotype x cell-type interaction (F 1,29 = 0.003, p = 0.95); Genotype main effect (F 1,29 = 9.32, * p < 0.01); Cell-type main effect (F 1,29 = 0.51, p = 0.48). c AMPAR/NMDAR ratios at synapses from PFC-to-DRN 5-HT neurons (left) and non-5-HT neurons (right) in control (5-HT: n = 10 cells/4 animals; non-5-HT: n = 7 cells/3 animals), and SERT-KO (5-HT: n = 11 cells/3 animals; non-5-HT: n = 6 cells; 3 animals). The AMPAR responses were calculated at the peak of −50 mV, whereas NMDAR responses were determined at + 40 mV, 50 ms after stimulation. Top: example traces; bottom: bar graphs. Two-ways ANOVA: Genotype x Cell-type interaction (F 1,30 = 0.007, p = 0.94); Genotype main effect (F 1,30 = 0.16, p = 0.69); Cell-type main effect (F 1,30 = 4.51, p < 0.05). Blue bars indicate blue light stimulation. Error bars represent SEM

Article Snippet: Subsequently, after a few hours of fixation in 4% PFA at 4 °C, brain slices were processed for immunohistochemistry using a rabbit polyclonal antibody against TPH2 (1:2000, Novus Biologicals, NB100-74555) to identify 5-HT neurons.

Techniques: Functional Assay, Injection, Control, Expressing, Patch Clamp, Immunolabeling

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin fragments are differentially localized in neurons. ( A ) WB of mouse whole brain protein samples with menin C-terminal ((C); left panel) and N-terminal ((N); right panel) epitope antibodies (n = 3 each, representative blots), depicting full length menin (black arrow), as well as N-terminal (light grey arrow) and C-terminal (dark grey arrow) menin proteolytic fragments. ( B ) WB of subcellular fractions from mouse brain protein samples with menin C-terminal ((C); top and middle panels) and N-terminal ((N); bottom panel) epitope antibodies (n = 6, representative blots). N denotes nuclear fraction, C denotes cytoplasmic fraction, S denotes synaptic fraction. Menin localizes to the nucleus, the C-menin fragment localizes to synaptic membranes, and the N-menin fragment localizes to the cytoplasm. ( C ) As in ( B ), the markers histone H3 (HH3; nuclear marker, top panel), β-tubulin (TUB; cytoplasmic marker, middle panel), and synaptophysin (SYP; synaptic marker, bottom panel), are shown to verify the subcellular fractions. ( D ) ICC localization of menin in hippocampal cultures at DIV 7 (n = 13 images, 4 independent samples, representative image), with N-terminal (i) and C-terminal (ii) epitope antibodies, and the nuclear stain DAPI (iii), (iv) shows merged channels. α-N-terminal menin signals are restricted to the nuclear and perinuclear region, α-C-terminal menin signals also exhibit punctate localization along neurites. Scale bar, 50 μm. See also Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Marker, Staining

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin is cleaved by calpain. ( A ) ICC localization of menin in hippocampal cultures at DIV 7, cultured in the presence of 0.1% DMSO vehicle control (i–v) or 20 μM PD150606 (vi–x), a cell permeable calpain inhibitor (n = 9 images, 3 independent samples each, representative images). Cells were labeled with the nuclear stain DAPI (i,vi), menin N-terminal (ii,vii) and C-terminal (iii,viii) epitope antibodies, and a neurofilament antibody (iv,ix), (v,x) shows merged channels. Scale bar, 50 μm. ( B ) Enlarged region of interest (ROI) depicting neurite structure from the boxed region shown in Av (i–iii) and Ax (iv–vi). Scale bar, 10 μm. ( C , D ) Summary data, fluorescence intensity of the nuclear α-N-menin signal was unaffected ( C ; ROIs: 0.1% DMSO n = 67, 20 μM PD150606 n = 59), whereas the neurite α-C-menin signal was reduced upon calpain inhibition ( D ; ROIs: n = 27 each). ***Statistical significance (independent t-test), P < 0.001. See also Table and Figs and .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Cell Culture, Control, Labeling, Staining, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: The C-terminal menin fragment colocalizes with α7 subunit-containing nAChRs at presynaptic terminals. ( A ) Super resolution image of a synaptic ROI at DIV 7 (n = 9 images, 2 independent samples, representative image), cells were labeled with α-C-terminal menin (i), α-bungarotoxin (ii; α7-nAChR), and α-synaptotagmin (iii; presynaptic marker), (iv) shows merged channels. ( B ) As in ( A ), only labeled with α-PSD-95 (iii; postsynaptic marker) (n = 10 images, 2 independent samples, representative image). Arrowheads, extrasynaptic colocalization of C-menin and α7-nAChRs; arrows, synaptic colocalization with synaptotagmin ( A ) or PSD-95 ( B ). Scale bar, 2 μm. ( C ) Summary data, incidence of colocalization of synaptotagmin (SYT; presynaptic), PSD-95 (postsynaptic), and α-bungarotoxin (BTX; α7-nAChR) puncta with C-menin. Asterisks, statistical significance (Kruskal-Wallis test); ** P < 0.01. *** P < 0.001. See also Table and Fig. .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Labeling, Marker

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin mediates subunit-specific transcriptional regulation of nAChR α5. ( A ) Live cell phase contrast ( i-iii ) and GFP fluorescence ( iv-vi ) images of untreated control (i,iv), NTC shRNA (ii,v), and MEN1 shRNA (iii,vi) lentivirus transduced hippocampal cultures at DIV 7 (n = 18 images, 6 independent samples each, representative images). Scale bar, 100 μm. ( B ) Summary data, fold change gene expression in hippocampal cultures at DIV 7, relative to untreated control, determined by qPCR (n = 6, 2 independent experiments each, triplicate replicates). MEN1 knockdown reduces nAChR α5 expression. ND, qPCR signal not detected. *Statistical significance (pair wise fixed reallocation randomization test), P < 0.05–0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Fluorescence, Control, shRNA, Gene Expression, Knockdown, Expressing

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nAChR α5 subunit expression. ( A ) ICC characterization of menin and nAChR protein expression in neuronal soma from NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures (n ≥ 4 images, ≥2 independent samples; see Table ; representative images, DIV 7). Untransduced neurons were GFP negative (−) and transduced neurons were GFP positive (+). The expression of menin was determined with C-terminal (i,v) and N-terminal (ii,vi) epitope antibodies, and nAChRs with a nAChR α5 antibody (iii,vii), or fluorophore-tagged α-BTX (iv,viii; α7-nAChR). ICC labels are shown in a (left panels), and GFP fluorescence is shown in b (right panels). Scale bars, 20 μm. ( B–E ) Summary data, normalized somal fluorescence intensity of α-C-terminal menin ( B ), α-N-terminal menin ( C ), α-nAChR α5 ( D ), and α-BTX ( E ), in GFP + neurons relative to GFP- neurons at DIV 3–14 (ROIs: n ≥ 12 each; see Table ). Dashed lines represent a 1:1 ratio indicating no change. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Expressing, shRNA, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: C-menin mediates neurite localization of α7 subunit-containing nAChRs. ( A , B ) ICC characterization of the neurite localization of C-menin and α7-nAChRs in NTC shRNA and MEN1 shRNA lentivirus transduced hippocampal cultures ( A ; n = 18 images, 5 independent samples each; representative images, DIV 7) or hippocampal cultures maintained in the presence of 0.1% DMSO or 20 μM PD150606 ( B ; n = 12 images, 2 independent samples each; representative images, DIV 7). Cells were labeled with the menin C-terminal epitope antibody (i-ii-a) or fluorophore-tagged α-BTX (iii-iv-a), and neurites were visualized with GFP ( A i-iv-b) or neurofilament ( B i-iv-b) antibodies, (i-iv-c) shows merged channels. Scale bars, 20 μm. ( C , D ) Summary data, the α-C-menin signal in neurites was reduced upon MEN1 knockdown ( C ), which coincides with a reduction of the neurite α-BTX signal ( D ). ( E ) Summary data, reduction of C-menin abundance by calpain inhibition (see Fig. ) coincides with a reduction of the neurite α-BTX signal (ROIs: n ≥ 36 each; see Table ), indicating that the synaptic targeting of α7-nAChRs requires the menin C-terminal fragment. Asterisks, statistical significance (Mann-Whitney U test); * P < 0.05. *** P < 0.001.

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: shRNA, Labeling, Knockdown, Inhibition, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces the presynaptic clustering of α7 subunit-containing nAChRs. ( A , B ) Super resolution images of C-menin, α-BTX, and SYT puncta in GFP+ synaptic ROIs from NTC shRNA ( A ) and MEN1 shRNA ( B ) lentivirus transduced hippocampal cultures at DIV 7 (n ≥ 10 images, 2 independent samples each, representative images). Cells were labeled with C-terminal menin and SYT antibodies (i–v; NTC: n = 16, MEN1 : n = 14), fluorophore-tagged α-BTX and a SYT antibody (vi–x; NTC: n = 16, MEN1 : n = 15), or α-BTX and a C-terminal menin antibody (xi–xv; NTC: n = 11, MEN1 : n = 10). (i,vi,xi) shows GFP fluorescence, (ii,xiii) shows α-C-terminal menin fluorescence, (vii,xii) shows α-BTX fluorescence, (iii,viii) shows α-SYT fluorescence, (iv,ix,xiv) shows SYT/C-menin/α-BTX merged channels, (v,x,xv) shows channels merged with GFP. Scale bar, 2 μm. ( C ) Summary data, mean number of α-C-terminal menin, α-BTX, and α-SYT puncta in super resolution images. ( D ) Summary data, incidence of colocalization of SYT/C-menin/α-BTX puncta in super resolution images. Asterisks, statistical significance (independent t-test or Mann-Whitney U test); * P < 0.05. ** P < 0.01. *** P < 0.001. See also Table .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, shRNA, Labeling, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Tumor suppressor menin is required for subunit-specific nAChR α5 transcription and nAChR-dependent presynaptic facilitation in cultured mouse hippocampal neurons

doi: 10.1038/s41598-017-01825-x

Figure Lengend Snippet: Menin knockdown reduces nicotine-induced presynaptic facilitation. ( A , B ) Representative patch clamp traces recorded from GFP+ hippocampal pyramidal neurons at DIV 10–14 (n ≥ 15, 3 independent experiments, representative traces). The mean frequency and amplitude of mEPSCs was analyzed before (Pre-nicotine, 10 s; top trace) and after (Post-nicotine, 10 s; bottom trace) the nicotine pulse (10 μM, 250 ms, 10 PSI; not shown). nAChR-mediated increase in mEPSC frequency (e.g. arrow) is observed in a GFP+ neuron from NTC-shRNA expressing cultures ( A ), but is absent in a GFP+ neuron from MEN1 -shRNA expressing cultures ( B ). ( C ) Summary data, incidence of nicotine-induced presynaptic facilitation in neurons from untreated control (n = 13/19), NTC shRNA (n = 11/15; GFP+) and MEN1 shRNA (n = 5/17; GFP+) transduced hippocampal cultures (post/pre-nicotine relative mEPSC frequency ≥1 ± SEM; see Fig. and Table ). *Statistical significance (Chi-squared test), P < 0.05. ( D ) Summary data, mean frequency of mEPSCs before (pre) and after (post) the nicotine pulse in the population of neurons from untreated control (n = 13), NTC shRNA (n = 11; GFP+) and MEN1 shRNA (n = 5; GFP+) transduced cultures that exhibit nicotine-induced presynaptic facilitation. ***Statistical significance (paired t-test) , P < 0.001. ( E ) Summary data, mean amplitude of mEPSCs, as in ( D ). See also Tables – and Figs – .

Article Snippet: Blocking and antibody incubations of WB PVDF membranes were performed with 5% skim milk powder + 0.1% Tween-20 in 1 × PBS for 1 h at room temperature or overnight at 4 °C (menin C-terminal epitope [Bethyl Laboratories, A300-105A, 1:2000]; menin N-terminal epitope [Santa Cruz Biotechnology, sc-374371, 1:2000]; histone H3 [Millipore, 06-599, 1:2000]; β-tubulin [Sigma-Aldrich, T0198, 1:2000]; synaptophysin [AbCam, ab52636, 1:2000]; IRDye-800CW conjugated α-mouse or α-rabbit IgG [Li-Cor biosciences, 925-32210, 925-32211, 1:5000]).

Techniques: Knockdown, Patch Clamp, shRNA, Expressing, Control

Journal: Journal of Medicinal Chemistry

Article Title: Utilizing Structures of CYP2D6 and BACE1 Complexes To Reduce Risk of Drug–Drug Interactions with a Novel Series of Centrally Efficacious BACE1 Inhibitors

doi: 10.1021/acs.jmedchem.5b00191

Figure Lengend Snippet: Effect of log D and p K a on hERG IC 50 . (A) Diverse Pfizer set of 2044 compounds. (B) Set of 169 BACE compounds from property space I and II. Red, hERG IC 50 < 10 μM; blue, hERG IC 50 > 10 μM. Total count per bin is highlighted in the center of the pie.

Article Snippet: Our analysis of a diverse set of over 2000 Pfizer compounds, profiled in the same hERG patch clamp assay, confirmed that reducing log D and lowering basic p K a were important factors in reducing binding to the hERG channel (Figure A).

Techniques: