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Formin1 mediates the induction of dendritogenesis and synaptogenesis by neurogenin3 in mouse hippocampal neurons.

Simon-Areces J, Dopazo A, Dettenhofer M, Rodriguez-Tebar A, Garcia-Segura LM, Arevalo MA - PLoS ONE (2011)

Bottom Line: One of such genes is Fmn1, whose protein, Formin1, is associated with actin and microtubule cytoskeleton.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroactive Steroids, Instituto Cajal, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.

ABSTRACT
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.

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Effects of Fmn1-Ib overexpression on the morphology and synaptology of cultured hippocampal neurons.Cells were tranfected at 3 DIV with an EGFP (A and C) or EGFP-Fmn1-Ib (B and D) expressing C2 vector and immunostained with antibodies against GFP and VGlut1 or VGAT. (A–D) Representative immunofluorescence images of neurons marked in green for GFP and with its synaptic contacts marked in red. Scale bar, 25 µm. Lower panels show the boxed regions at higher magnification. (E) Number of primary dendrites. (F) Counts of VGlut1 immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 50 µm and centered in the neuronal soma. (G) Counts of VGAT immunoreactive terminals in contact with a neuron per ROI. Typically 60–75 neurons were evaluated in each condition (n = 3). (H) Ratio of excitatory/inhibitory (E/I) synaptic terminal number. Data are mean ± SEM and significance levels were determined using a Student t-test; ** p<0.01, *** p<0.001 versus EGFP expressing neurons values.
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pone-0021825-g003: Effects of Fmn1-Ib overexpression on the morphology and synaptology of cultured hippocampal neurons.Cells were tranfected at 3 DIV with an EGFP (A and C) or EGFP-Fmn1-Ib (B and D) expressing C2 vector and immunostained with antibodies against GFP and VGlut1 or VGAT. (A–D) Representative immunofluorescence images of neurons marked in green for GFP and with its synaptic contacts marked in red. Scale bar, 25 µm. Lower panels show the boxed regions at higher magnification. (E) Number of primary dendrites. (F) Counts of VGlut1 immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 50 µm and centered in the neuronal soma. (G) Counts of VGAT immunoreactive terminals in contact with a neuron per ROI. Typically 60–75 neurons were evaluated in each condition (n = 3). (H) Ratio of excitatory/inhibitory (E/I) synaptic terminal number. Data are mean ± SEM and significance levels were determined using a Student t-test; ** p<0.01, *** p<0.001 versus EGFP expressing neurons values.

Mentions: We have previously reported that under conditions of intermediate cell density, overexpression of Ngn3 stimulates the sprouting of new dendrites in cultured hippocampal neurons [6]. Conversely, addition of NGF to cultures induces the down-regulation of Ngn3 mRNA levels and hence hippocampal neurons sprout fewer primary and secondary dendrites. Since Ngn3 enhances the expression of Fmn1, we decided to test the effects of Fmn1 overexpression on hippocampal neuronal development. Hippocampal neuronal cultures were transfected with vectors expressing EGFP and EGFP-Fmn1-Ib. Specimen images of transfected cells are presented in Figure 3, A–D. As expected, the protein encoded by EGFP-Fmn1-Ib was not found in the nuclei. The quantification of the results showed that overexpression of Fmn1-Ib resulted in a clear stimulation of dendrite initiation (Figure 3E). The number of primary dendrites was increased by 50% versus control levels by 16 h after transfection with EGFP-Fmn1-Ib.


Formin1 mediates the induction of dendritogenesis and synaptogenesis by neurogenin3 in mouse hippocampal neurons.

Simon-Areces J, Dopazo A, Dettenhofer M, Rodriguez-Tebar A, Garcia-Segura LM, Arevalo MA - PLoS ONE (2011)

Effects of Fmn1-Ib overexpression on the morphology and synaptology of cultured hippocampal neurons.Cells were tranfected at 3 DIV with an EGFP (A and C) or EGFP-Fmn1-Ib (B and D) expressing C2 vector and immunostained with antibodies against GFP and VGlut1 or VGAT. (A–D) Representative immunofluorescence images of neurons marked in green for GFP and with its synaptic contacts marked in red. Scale bar, 25 µm. Lower panels show the boxed regions at higher magnification. (E) Number of primary dendrites. (F) Counts of VGlut1 immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 50 µm and centered in the neuronal soma. (G) Counts of VGAT immunoreactive terminals in contact with a neuron per ROI. Typically 60–75 neurons were evaluated in each condition (n = 3). (H) Ratio of excitatory/inhibitory (E/I) synaptic terminal number. Data are mean ± SEM and significance levels were determined using a Student t-test; ** p<0.01, *** p<0.001 versus EGFP expressing neurons values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139584&req=5

pone-0021825-g003: Effects of Fmn1-Ib overexpression on the morphology and synaptology of cultured hippocampal neurons.Cells were tranfected at 3 DIV with an EGFP (A and C) or EGFP-Fmn1-Ib (B and D) expressing C2 vector and immunostained with antibodies against GFP and VGlut1 or VGAT. (A–D) Representative immunofluorescence images of neurons marked in green for GFP and with its synaptic contacts marked in red. Scale bar, 25 µm. Lower panels show the boxed regions at higher magnification. (E) Number of primary dendrites. (F) Counts of VGlut1 immunoreactive terminals in contact with a neuron within a circular region of interest (ROI) with a diameter of 50 µm and centered in the neuronal soma. (G) Counts of VGAT immunoreactive terminals in contact with a neuron per ROI. Typically 60–75 neurons were evaluated in each condition (n = 3). (H) Ratio of excitatory/inhibitory (E/I) synaptic terminal number. Data are mean ± SEM and significance levels were determined using a Student t-test; ** p<0.01, *** p<0.001 versus EGFP expressing neurons values.
Mentions: We have previously reported that under conditions of intermediate cell density, overexpression of Ngn3 stimulates the sprouting of new dendrites in cultured hippocampal neurons [6]. Conversely, addition of NGF to cultures induces the down-regulation of Ngn3 mRNA levels and hence hippocampal neurons sprout fewer primary and secondary dendrites. Since Ngn3 enhances the expression of Fmn1, we decided to test the effects of Fmn1 overexpression on hippocampal neuronal development. Hippocampal neuronal cultures were transfected with vectors expressing EGFP and EGFP-Fmn1-Ib. Specimen images of transfected cells are presented in Figure 3, A–D. As expected, the protein encoded by EGFP-Fmn1-Ib was not found in the nuclei. The quantification of the results showed that overexpression of Fmn1-Ib resulted in a clear stimulation of dendrite initiation (Figure 3E). The number of primary dendrites was increased by 50% versus control levels by 16 h after transfection with EGFP-Fmn1-Ib.

Bottom Line: One of such genes is Fmn1, whose protein, Formin1, is associated with actin and microtubule cytoskeleton.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neuroactive Steroids, Instituto Cajal, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus