9 research outputs found
Construction of Polyheterocyclic Benzopyran Library with Diverse Core Skeletons through Diversity-Oriented Synthesis Pathway: Part II
As a continuation of our previous report (<i>J.
Comb. Chem.</i> <b>2010</b>, <i>12</i>, 548–558), we accomplished the
diversity-oriented synthesis of polyheterocyclic small-molecule library
with privileged benzopyran substructure. To ensure the synthetic efficiency,
we utilized the solid-phase parallel platform and the fluorous-tag-based
solution-phase parallel platform to construct a 284-member polyheterocyclic
library with six distinct core skeletons with an average purity of
87% on a scale of 5–10 mg. This library was designed to maximize
the skeletal diversity with discrete core skeletons in three-dimensional
space and the combinatorial diversity with four different benzopyranyl
starting materials and various building blocks. Together with our
reported benzopyranyl library, we completed the construction of polyheterocyclic
benzopyran library with 11 unique scaffolds and their molecular diversity
was visualized in chemical space using principle component analysis
(PCA)
CREB is activated by BDNF-stimulation, and required for BDNF-dependent spinogenesis.
<p>DIV6 cultured hippocampal neurons were transfected with m-RFP-βActin ± empty vector (Control and BDNF), ± ACREB, ± sh-CREB, or ± caCREB, and then treated ±50 ng/mL BDNF from DIV7–12. Cultures were then used for electrophysiological recordings, or fixed, mounted, immunostained, and imaged. A) Representative images and quantification of dendritic spine type and filopodia density is shown, with total spine number as the combination of mushroom and stubby spines. Inset panels) DIV6 cultured hippocampal neurons were treated ±50 ng/mL BDNF for 20 minutes ±1 hour pre-treatment with 20 µM U0–126, lysed and then analyzed by Western Blot, using anti-phospho-ERK, anti-phospho-CREB, and anti-ERK2 (loading control) antibodies. B) Representative images of neurons immunostained using anti-VGlut1 and anti-Syanapsin1 antibodies. C) Quantification of percent co-localization of presynaptic markers with dendritic spine heads (50–100 spines measured on 10–12 hippocampal neurons per condition in two experiments). D) Quantification of average spine head width (50–100 spines measured on 10–12 neurons per condition in two experiments). E) Representative traces of mEPSCs recorded from control or BDNF treated neurons at DIV12 following ± BDNF stimulation from DIV7–12 (50–80 neurons in 6 experiments). F) Average frequency of mEPSCs. G) mEPSC cumulative distribution of inter-event intervals. (± SEM, Statistical analyses utilized Student’s t-test and ANOVA with Tukey’s post-test, *p<0.001 compared to control, #p<0.001 compared to BDNF).</p
RhoA activity inhibits BDNF-induced dendritic spine formation.
<p>A) DIV6 cultured hippocampal neurons were transfected with m-RFP-βActin ± empty vector (Control and BDNF), ± caRhoA, ± dnRhoA, ± sh-RhoA, ± sh-p190GAP, ± p190GAP, then treated ±50 ng/mL BDNF on DIV7 until fixed on DIV12. Dendrites were imaged, and two to three different sections of dendrite per neuron (>24neurons per condition from 2 or more independent experiments were analyzed). Quantification of dendritic spine type and filopodia density is shown, with total spine number representing the combination of mushroom and stubby spines. B) Organotypic hippocampal slice cultures were transfected on DIV2 with Tomato (TFP) (Control and BDNF), ± caRhoA, ± dnRhoA, ± sh-RhoA, then treated ±50 ng/mL BDNF on DIV4 until fixed on DIV6. Dendrites of CA1 pyramidal neurons were imaged, and a single dendrite was analyzed per neuron (20–50 neurons/condition from 3 independent cultures). C) Dissociated hippocampal neurons were transfected on DIV6 with m-RFP-βActin and empty vector, myc-RhoA, myc-RhoA+sh-RhoA, myc-p190GAP, myc-p190GAP+sh-p190GAP. Neurons were fixed on DIV12, and immunostained using anti-myc antibody, and imaged with 60X lens. (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test, *p<0.001 compared to control, #p<0.001 compared to BDNF).</p
The RhoA inhibitors Rnd3 and Par6C are BDNF-regulated CREB target genes.
<p>(A and B) UCSC Genome browser tracks depicting CREB ChIP-Seq peaks and CRE motifs at the Par6C/pard6a (A) and Rnd3 (B) genes. The CRE motifs in the Par6C gene are the canonical motif (TGACG) while all three of the CRE motifs in the Rnd3 gene are the non-canonical CRE (TGGCG). (C) Mouse hippocampal neuron chromatin was immunoprecipitated with the indicated antibodies. Primers to the Fos, Par6C, and Rnd3 ChIP-Seq peaks were used to assess occupancy of CREB at these loci. GAPDH represents a control locus not predicted to be occupied by CREB. (IgG, n = 2; CREB, n = 4; SEM). (D and E) Hippocampal neurons were transfected with empty vector (E.V.) or ACREB DNAs, and treated on DIV6±50 ng/mL BDNF for 1 or 2 hrs. RNA was reverse transcribed and relative cDNA levels of Par6C (D) and Rnd3 (E) were assessed by real-time PCR. Relative cDNA levels were normalized to relative GAPDH cDNA levels also measured by real time PCR. (n = 4; SEM). (F–G) DIV6 cultured hippocampal neurons were treated ±50 ng/mL BDNF for 4 hours ±1 hour pre-treatment with 20 µM U0126, lysed, and then analyzed by Western Blot for Par6C and Rnd3 with ERK2 used as a loading control. Changes in protein band intensity were determined by densitometry using image J. Graph depicts percent change in band intensity relative to control, and normalized to ERK2 loading control band intensity, representing 3 separate experiments. Data is presented as % of control ± SEM. (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test, *p<0.01 compared to control).</p
Par6C and Rnd3 are essential mediators of BDNF-induced spinogenesis in organotypic slice cultures.
<p>Organotypic hippocampal slice cultures were transfected on DIV2 with Tomato (TFP) (Control and BDNF), A) ± Par6C, ± Par6C+caRhoA, ± Par6C+sh-p190GAP ± sh-Par6C. B) ± Rnd3, ± Rnd3+caRhoA, Rnd3+sh-p190GAP, ± si-Rnd3, and then stimulated with ±50 ng/mL BDNF on DIV4 until fixed on DIV6. Dendrites of CA1 pyramidal neurons were imaged, and a single dendrite was analyzed per neuron (30–60 neurons/condition in 3 separate experiments). (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test (*p<0.001 compared to control, #p<0.001 compared to BDNF, ¥p<0.001 compared to Par6 or Rnd3). E) Model of BDNF- and CREB-dependent synaptogenesis.</p
Par6C is an essential mediator of BDNF-induced synaptogenesis.
<p>DIV6 cultured hippocampal neurons were transfected with mRFP-βActin ± empty vector (Control and BDNF), ± Par6C, ± Par6C+caRhoA, ± sh-Par6C, ± sh-p190GAP, ± sh-p190GAP+Par6C, ± p190GAP, ± p190GAP+shPart6C, and then treated ±50 ng/mL BDNF on DIV7 until fixed on DIV12. Cultures were then used for electrophysiological recordings, or fixed, mounted, immunostained, and imaged. A) Representative images and quantification of dendritic spine type and filopodia density is shown, with total spine number representing the combination of mushroom and stubby spines (2–3 different dendritic sections (>50 µm) on 24–60 neurons per condition were analyzed in 2 or more experiments). B) Representative traces of mEPSCs recorded from hippocampal neurons. C) Average frequencies of mEPSCs relative to control (20–40 neurons in 2–4 experiments). D) Average spine head width. E) Representative images of neurons immunostained using anti-VGlut1 and anti-Syanapsin1 antibodies. F) Quantification of percent co-localization of presynaptic markers with dendritic spine heads. G) HEK cells transfected with myc-Par6C ± sh-Par6C, and cell lysates analyzed using Western Blot and stained using anti-Par6C and anti-ERK2 antibodies. Representative images of neurons transfected on DIV6 with mRFP-βActin ± empty vector, ± Par6C-myc, or ± Par6C-myc+sh-Par6C. On DIV12 neurons were fixed and immunostained using anti-myc antibody, and imaged with 60X lens. (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test, *p<0.001 compared to control, #p<0.001 compared to BDNF, ¥p<0.001 compared to Par6C).</p
Rnd3 is an essential mediator of BDNF-induced synaptogenesis.
<p>DIV6 cultured hippocampal neurons were transfected with m-RFP-βActin ± empty vector (Control and BDNF), ± Rnd3, ± Rnd3+caRhoA, ± si-Rnd3, ± sh-p190GAP, ± sh-p190GAP+Rnd3, ± p190GAP, ± p190GAP+siRnd3 and then treated ±50 ng/mL BDNF on DIV7 until fixed on DIV12. Cultures were then used for electrophysiological recordings, or fixed, mounted, immunostained, and imaged. A) Representative images and quantification of dendritic spine type and filopodia density is shown, with total spine number representing the combination of mushroom and stubby spines (2–3 different dendritic sections (>50 µm) on 24–60 neurons per condition were analyzed in 2 or more experiments). B) Representative traces of mEPSCs recorded from hippocampal neurons. C) Average frequencies of mEPSCs relative to control (20–35 neurons in 2–4 experiments). D) Average spine head width. E) Representative images of neurons immunostained using anti-VGlut1 and anti-Syanapsin1 antibodies. F) Quantification of percent co-localization of presynaptic markers with dendritic spine heads. G) HEK cells transfected with myc-Rnd3± si-Rnd3, and cell lysates analyzed using Western Blot, and stained using anti-Rnd3 and anti-ERK2 antibodies. Representative images of neurons transfected on DIV6 with mRFP βActin ± empty vector, ± myc-Rnd3, or ± myc-Rnd3+si-Rnd3. On DIV12 neurons were fixed and immunostained using anti-myc antibody, and imaged with 60X lens. (± SEM, Statistical analyses utilized ANOVA and Tukey’s post-test, *p<0.001 compared to control, #p<0.001 compared to BDNF, ¥p<0.001 compared to Rnd3).</p
CREB ChIP-Seq identifies non-canonical CRE motif.
<p>(A and B) UCSC Genome browser tracks depicting CREB ChIP-Seq peaks and CRE motifs at known targets BDNF and JunD. (C) Hippocampal neuron CREB ChIP material was interrogated by real-time PCR using primers designed to a region within ∼200 bp of the indicated peak centroid (weighted average of positions). All ChIP-Seq peaks were confirmed to be occupied by CREB (n = 3, SEM). 18s and GAPDH are negative controls while c-fos (green bar) is a positive control. (D and E) ChIP-Seq tag density relative to the top third of expressed genes in hippocampal neurons (D) and the bottom third (E). Expression levels were determined by sorting hippocampal RNA-Seq data by normalized tag counts within RefSeq gene exons. All Ref-Seq gene lengths were scaled to 1. (F) <i>De novo</i> motif algorithms identify canonical and non-canonical motif in CREB ChIP-Seq data. (G) Pie chart depicts presence of CRE and non-canonical motif within 2kb of a ChIP-Seq peak. (H and I) Visualization of electrostatic interactions between DNA phosphate backbone and CREB bZIP domain for the consensus somatostatin CRE (H) and a modeled TGGCG mutated CRE (I). Charged amino acids making phosphate contacts are labeled. Carbon atoms are white, oxygen atoms are red, nitrogen atoms are blue and phosphate atoms are yellow. (J and K) Accumulation plots depicting the percentage of indicated motifs present in all CREB ChIP-Seq peaks (500 bp window surrounding center of mass). Offset denotes a control region 2 kb upstream of ChIP-Seq loci. Random indicates randomized motif positions. The grey lines depict motifs in which a single base has been changed to T. (L and M) Hippocampal neurons were transfected with the indicated luciferase reporter constructs and treated with forskolin (L) or co-transfected with constitutively active CREB (ca-CREB) (M). (N) PC12 cells were transfected with the indicated reporter constructs and subjected to ChIP for CREB. Primers directed against the reporter enhancer site were used to assess recruitment of CREB via real-time PCR.</p
The non-canonical CRE is associated with CREB occupancy and CREB-responsiveness of endogenous genes.
<p>(A and B) Histograms depict spatial accumulation of mouse hippocampal CREB ChIP-Seq peaks relative to all genomic occurrences of the canonical CRE (TGACG) and the non-canonical CRE (TGGCG). Randomized peaks over the same genomic extent are depicted in grey. (C-E) UCSC genome browser tracks show hippocampal CREB ChIP-Seq (CREB hip), embryonic cortical neuron CREB ChIP-Seq (CREB embryonic) data relative to RefSeq genes, non-canonical CRE motifs (red), and CpG islands (green). Rat CREB SACO data mapped to the mouse genome is also depicted with purple bars denoting SACO cluster extent (CREB SACO). The depicted loci did not contain the canonical CRE motif. (F) Chromatin from rat and mouse hippocampal neurons was immunoprecipitated with the indicated antibodies. Real-time PCR with primers directed against the ChIP-Seq peak (mouse) or the orthologous rat locus (rat) were used to assess CREB occupancy (n = 3; SEM). Significance was assessed using the Storey-adjusted Fisher exact test (FDR-adjusted p). (G) All RefSeq genes whose annotated transcriptional start is within 1 kb of a hippocampal neuron ChiP-Seq peak were selected for gene ontology analyses. Significance was assessed using the Storey-adjusted Fisher exact test (FDR-adjusted p).</p