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A genome-wide screen of CREB occupancy identifies the RhoA inhibitors Par6C and Rnd3 as regulators of BDNF-induced synaptogenesis.

Lesiak A, Pelz C, Ando H, Zhu M, Davare M, Lambert TJ, Hansen KF, Obrietan K, Appleyard SM, Impey S, Wayman GA - PLoS ONE (2013)

Bottom Line: Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region.Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis.Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Program in Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis.

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Par6C is an essential mediator of BDNF-induced synaptogenesis.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).
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pone-0064658-g006: Par6C is an essential mediator of BDNF-induced synaptogenesis.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).

Mentions: Par6C has been shown to stimulate spine formation in hippocampal neurons by controlling p190RhoGAP-mediated inhibition of RhoA [20]–[25]; however, relatively little is known about the mechanisms that regulate Par6C expression and function. Overexpression of Par6C increases the density of both mushroom and stubby spines (Figure 6A). Co-expression of caRhoA with Par6C blocked Par6C-stimulated spinogenesis (Figure 6A). sh-RNA-mediated repression of Par6C expression (Figure 6G) decreased spine densities under basal conditions, and blocked BDNF-induced spinogenesis (Figure 6A). Because, sh-RNA-mediated knockdown can have off target effects we confirmed that expression of an sh-RNA construct targeting a distinct region of the Par6C mRNA transcript also inhibited BDNF-induced spine formation. Additionally, we found that neither a non-scrambled control sh-RNA, nor a sh-RNA that inhibits expression of Rac3 (a RhoGTPase actin regulator not required for spine formation [19], [66]) prevent BDNF-induced increases in dendritic spine density (Figure S1). p190RhoGAP over-expression was effective in stimulating a significant increase in dendritic spine density, and effectively rescued the inhibition of spine formation induced by Par6C suppression (Figure 6A). Furthermore, shRNA-mediated suppression of p190GAP significantly decreased levels of dendritic spine density below basal levels with and without co-expression of Par6C (Figure 6A). Par6C, like BDNF-treatment, significantly increased average spine head width above control levels, while sh-Par6C significantly decreased average spine head width below control levels, with and without BDNF-treatment (Figure 6D). Additionally, both VGlut1 and Synapsin1 were highly co-localized (∼80–90%) with dendritic spines in control, BDNF, and Par6C conditions (Figure 6E and F). Furthermore, expression of Par6C increased the frequency of mEPSCs approximately 2-fold over control levels, close to the levels seen following BDNF stimulation (Figure 6B–C). In contrast, targeted knockdown of Par6C had no significant effect on mEPSC frequency compared to control, but blocked the effect of BDNF on mEPSC frequency. Neither increasing nor decreasing the expression of Par6C affected mEPSC amplitude, rise time, or decay time (data not shown).


A genome-wide screen of CREB occupancy identifies the RhoA inhibitors Par6C and Rnd3 as regulators of BDNF-induced synaptogenesis.

Lesiak A, Pelz C, Ando H, Zhu M, Davare M, Lambert TJ, Hansen KF, Obrietan K, Appleyard SM, Impey S, Wayman GA - PLoS ONE (2013)

Par6C is an essential mediator of BDNF-induced synaptogenesis.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).
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pone-0064658-g006: Par6C is an essential mediator of BDNF-induced synaptogenesis.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).
Mentions: Par6C has been shown to stimulate spine formation in hippocampal neurons by controlling p190RhoGAP-mediated inhibition of RhoA [20]–[25]; however, relatively little is known about the mechanisms that regulate Par6C expression and function. Overexpression of Par6C increases the density of both mushroom and stubby spines (Figure 6A). Co-expression of caRhoA with Par6C blocked Par6C-stimulated spinogenesis (Figure 6A). sh-RNA-mediated repression of Par6C expression (Figure 6G) decreased spine densities under basal conditions, and blocked BDNF-induced spinogenesis (Figure 6A). Because, sh-RNA-mediated knockdown can have off target effects we confirmed that expression of an sh-RNA construct targeting a distinct region of the Par6C mRNA transcript also inhibited BDNF-induced spine formation. Additionally, we found that neither a non-scrambled control sh-RNA, nor a sh-RNA that inhibits expression of Rac3 (a RhoGTPase actin regulator not required for spine formation [19], [66]) prevent BDNF-induced increases in dendritic spine density (Figure S1). p190RhoGAP over-expression was effective in stimulating a significant increase in dendritic spine density, and effectively rescued the inhibition of spine formation induced by Par6C suppression (Figure 6A). Furthermore, shRNA-mediated suppression of p190GAP significantly decreased levels of dendritic spine density below basal levels with and without co-expression of Par6C (Figure 6A). Par6C, like BDNF-treatment, significantly increased average spine head width above control levels, while sh-Par6C significantly decreased average spine head width below control levels, with and without BDNF-treatment (Figure 6D). Additionally, both VGlut1 and Synapsin1 were highly co-localized (∼80–90%) with dendritic spines in control, BDNF, and Par6C conditions (Figure 6E and F). Furthermore, expression of Par6C increased the frequency of mEPSCs approximately 2-fold over control levels, close to the levels seen following BDNF stimulation (Figure 6B–C). In contrast, targeted knockdown of Par6C had no significant effect on mEPSC frequency compared to control, but blocked the effect of BDNF on mEPSC frequency. Neither increasing nor decreasing the expression of Par6C affected mEPSC amplitude, rise time, or decay time (data not shown).

Bottom Line: Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region.Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis.Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Program in Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis.

Show MeSH
Related in: MedlinePlus