Estradiol Meets Notch Signaling in Developing Neurons

The transmembrane receptor Notch, a master developmental regulator, controls gliogenesis, neurogenesis, and neurite development in the nervous system. Estradiol, acting as a hormonal signal or as a neurosteroid, also regulates these developmental processes. Here we review recent evidence indicating that estradiol and Notch signaling interact in developing hippocampal neurons by a mechanism involving the putative membrane receptor G protein-coupled receptor 30. This interaction is relevant for the control of neuronal differentiation, since the downregulation of Notch signaling by estradiol results in the upregulation of neurogenin 3, which in turn promotes dendritogenesis

Formin1 Mediates the Induction of Dendritogenesis and Synaptogenesis by Neurogenin3 in Mouse Hippocampal Neurons

Neurogenin3, a proneural transcription factor controlled by Notch receptor, has been recently shown to regulate dendritogenesis and synaptogenesis in mouse hippocampal neurons. However, little is known about the molecular mechanisms involved in these actions of Ngn3. We have used a microarray analysis to identify Ngn3 regulated genes related with cytoskeleton dynamics. One of such genes is Fmn1, whose protein, Formin1, is associated with actin and microtubule cytoskeleton. Overexpression of the Fmn1 isoform-Ib in cultured mouse hippocampal neurons induced an increase in the number of primary dendrites and in the number of glutamatergic synaptic inputs at 4 days in vitro. The same changes were provoked by overexpression of Ngn3. In addition downregulation of Fmn1 by the use of Fmn1-siRNAs impaired such morphological and synaptic changes induced by Ngn3 overexpression in neurons. These results reveal a previously unknown involvement of Formin1 in dendritogenesis and synaptogenesis and indicate that this protein is a key component of the Ngn3 signaling pathway that controls neuronal differentiation

Pharmacological perturbation of the cytoskeleton suggests association of Ngn3 to tubulin and microtubules.

<p>(<b>A</b>) Cultured neurons were untreated or treated with paclitaxel (Taxol; 20 µM) for 40 minutes; then cells were lysated and centrifuged. Proteins present in the precipitate, that includes microtubules and associated proteins (P) and supernatant (S) were analyzed by Western blotting. Ngn3 is enriched in the insoluble fractions and its concentration increases as polymerized tubulin does. (<b>B</b>) Embryonic mouse brains were homogenized and a high-speed precipitate was resuspended and divided in two. Aliquots were left untreated or treated with 20 µM paclitaxel plus 1 mM GTP for 40 minutes at room temperature. Microtubular fraction sedimented by centrifugation (P) and supernatant (S) were analyzed by Western blotting. (<b>C</b>) The supernatants (S) of control (C) and paclitaxel (T) treated aliquots were immunoprecipitated (IP) with anti-βIII-tubulin antibody (or IgG control) to determine the interaction of Ngn3 with soluble tubulin. Precipitates were analyzed by Western blotting with anti-Ngn3 antibody. Graphs show the quantification of densitometry. Error bars show the mean+s.e.m. of three experiments. Significance levels were determined for the data sets connected by horizontal lines using the Student’s t-test. * p<0.05, **p<0.01.</p

Primer sequences used for construction of EGFP-NES2 mutants.

<p>Primer sequences used for construction of EGFP-NES2 mutants.</p

Mutation of the NES2 (L135A) counteracts the effects of Ngn3 overexpression on neuronal morphology and synaptic inputs.

<p>(<b>A–C</b>) Hippocampal neuronal cultures were co-transfected with constructs encoding EGFP and full-length myc-tagged wild-type Ngn3 (myc-Ngn3), Ngn3 with leucine 135 mutated to alanine (myc-Ngn3-L135A) or empty vector expressing myc-tag as control. After 16 h, double immunostaining was performed using an anti-GFP antibody to visualize transfected neurons and an anti-synaptophysin I antibody to determine the morphology and the total number of synapses of the transfected neurons. (<b>D–F</b>) Lower panels show the boxed regions at higher magnification (<b>G</b>) Number of primary dendrites of the transfected neurons. (<b>H</b>) Counts of synaptophysin I immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 100 µm and centered in the neuronal soma. Data are mean+s.e.m. and significance levels were determined using ANOVA followed by the Bonferroni post hoc test; *** p<0.001 versus control neuron values and ### p<0.001 versus myc-Ngn3 expressing neuron values.</p

Point mutation in NES2 induces nuclear accumulation of full-length Ngn3.

<p>(<b>A–F</b>) Cultured neurons were co-transfected with constructs encoding EGFP and full-length myc-tagged wild-type Ngn3 (myc-Ngn3), Ngn3 with leucine 135 mutated to alanine (myc-Ngn3-L135A) or empty vector expressing myc-tag. After 16 h, double immunostaining was performed using an anti-myc antibody to determine subcellular localization of wild-type and mutated myc-Ngn3 (A-C) and an anti-GFP antibody to visualize neurons at full (D-F). (<b>G</b>) Quantification of the relative fluorescence in the cell nucleus versus the cytoplasm of neurons. Graphs show the results (mean+s.e.m.) of at least three experiments. Significance levels were determined using a Student’s t-test; *** p<0.001 versus values of neurons transfected with plasmid expressing myc-tag.</p

Double-stranded oligonucleotides used for the construction of wild-type EGFP-NES1 and EGFP-NES2 constructs.

<p>Double-stranded oligonucleotides used for the construction of wild-type EGFP-NES1 and EGFP-NES2 constructs.</p