Limits...
An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice.

Srivastava DP, Woolfrey KM, Jones KA, Anderson CT, Smith KR, Russell TA, Lee H, Yasvoina MV, Wokosin DL, Ozdinler PH, Shepherd GM, Penzes P - PLoS Biol. (2012)

Bottom Line: We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect.Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology.These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

Show MeSH

Related in: MedlinePlus

Epac2 regulates basal dendrite complexity in vivo.(A) Low magnification images of in utero electroporated layer 2/3 cortical neurons, expressing either control or Epac2-RNAi. Animals were electroporated at E16.5; coronal slices (50 µm) were made at P28. Red dashed rectangles indicate images represented in (B–D). (B) High magnification images of layer 2/3 cortical neurons outlined in (A). (C–D) Binary images (C) and “skeleton outline” of basal and apical arbors (D), separated into primary, secondary, and tertiary branches, of neurons shown in (B). (E) Quantification of dendrite numbers separated into total apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites. (F) Epac2 knockdown in vivo induced a selective reduction in basal dendrite length. (G) Quantification of dendrite branch numbers separated into basal/apical, primary/secondary/tertiary order branches. Epac2 knockdown in vivo induced a loss of basal dendrites, specifically driven by a loss of secondary and tertiary basal dendrites. (H) Epac2 knockdown in vivo selectively reduced secondary and tertiary basal dendrite lengths. *p<0.05, ***p<0.001; scale bars, 100 µm (A); 50 µm (B–D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383751&req=5

pbio-1001350-g001: Epac2 regulates basal dendrite complexity in vivo.(A) Low magnification images of in utero electroporated layer 2/3 cortical neurons, expressing either control or Epac2-RNAi. Animals were electroporated at E16.5; coronal slices (50 µm) were made at P28. Red dashed rectangles indicate images represented in (B–D). (B) High magnification images of layer 2/3 cortical neurons outlined in (A). (C–D) Binary images (C) and “skeleton outline” of basal and apical arbors (D), separated into primary, secondary, and tertiary branches, of neurons shown in (B). (E) Quantification of dendrite numbers separated into total apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites. (F) Epac2 knockdown in vivo induced a selective reduction in basal dendrite length. (G) Quantification of dendrite branch numbers separated into basal/apical, primary/secondary/tertiary order branches. Epac2 knockdown in vivo induced a loss of basal dendrites, specifically driven by a loss of secondary and tertiary basal dendrites. (H) Epac2 knockdown in vivo selectively reduced secondary and tertiary basal dendrite lengths. *p<0.05, ***p<0.001; scale bars, 100 µm (A); 50 µm (B–D).

Mentions: Epac2, an upstream regulator of Rap activity, is involved in regulating synapse morphology [22], but its role in regulating the architecture of the dendritic tree in cortical neurons is unknown. We first tested whether altered expression of Epac2 affected the maintenance of dendritic arbors in vivo, using in utero electroporation (IUEP) [30] to knock down protein expression (Figure S1A). Using a previously characterized RNA interference (RNAi) construct selective for Epac2 [22], we coinjected either Epac2-RNAi, or control (pGSuper), with pCAG-eGFP into the subventricular zone of E16.5 mouse embryos to specifically target layer 2/3 neurons. Electroporated constructs were allowed to express until brain harvesting and sectioning on postnatal day 28 (P28). In vivo knockdown of the Epac2 protein via Epac2-RNAi was confirmed at P28 by immunohistochemistry of cortical sections. Quantification of GFP-positive cells revealed that Epac2 was knocked down by ∼75% compared to GFP-negative cells (Figure S1B–C). Electroporated neurons were specifically found in layer 2/3 (Figure 1A). When we analyzed dendritic morphology by measuring apical and basal dendritic number and length in GFP-positive cells, we observed that long-term knockdown of Epac2 expression specifically reduced basal dendrites. Examination of basal arbors of layer 2/3 neurons from P28 mice expressing GFP and Epac2-RNAi (Figure 1B–F) revealed a ∼42% reduction in basal dendritic branches and a ∼54% reduction in basal dendritic length (p<0.001; Figure 1E–F) compared to cells from P28 mice expressing GFP and control construct. This effect on basal dendritic morphology was mediated by a ∼30% reduction in secondary and a ∼65% reduction in tertiary order basal branch number (p<0.05, 0.001; Figure 1G, Table S1A). Additionally, basal dendritic length was reduced by ∼24% in secondary branches and ∼55% in tertiary branches (p<0.05, 0.001; Figure 1H, Table S1B). The effect of Epac2 knockdown from E16.5 to P28 was specific for basal dendritic arbors, as Epac2-RNAi had no effect on apical dendrite branch number or length in these cells (Figure 1E–H, Table S1A–B).


An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice.

Srivastava DP, Woolfrey KM, Jones KA, Anderson CT, Smith KR, Russell TA, Lee H, Yasvoina MV, Wokosin DL, Ozdinler PH, Shepherd GM, Penzes P - PLoS Biol. (2012)

Epac2 regulates basal dendrite complexity in vivo.(A) Low magnification images of in utero electroporated layer 2/3 cortical neurons, expressing either control or Epac2-RNAi. Animals were electroporated at E16.5; coronal slices (50 µm) were made at P28. Red dashed rectangles indicate images represented in (B–D). (B) High magnification images of layer 2/3 cortical neurons outlined in (A). (C–D) Binary images (C) and “skeleton outline” of basal and apical arbors (D), separated into primary, secondary, and tertiary branches, of neurons shown in (B). (E) Quantification of dendrite numbers separated into total apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites. (F) Epac2 knockdown in vivo induced a selective reduction in basal dendrite length. (G) Quantification of dendrite branch numbers separated into basal/apical, primary/secondary/tertiary order branches. Epac2 knockdown in vivo induced a loss of basal dendrites, specifically driven by a loss of secondary and tertiary basal dendrites. (H) Epac2 knockdown in vivo selectively reduced secondary and tertiary basal dendrite lengths. *p<0.05, ***p<0.001; scale bars, 100 µm (A); 50 µm (B–D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383751&req=5

pbio-1001350-g001: Epac2 regulates basal dendrite complexity in vivo.(A) Low magnification images of in utero electroporated layer 2/3 cortical neurons, expressing either control or Epac2-RNAi. Animals were electroporated at E16.5; coronal slices (50 µm) were made at P28. Red dashed rectangles indicate images represented in (B–D). (B) High magnification images of layer 2/3 cortical neurons outlined in (A). (C–D) Binary images (C) and “skeleton outline” of basal and apical arbors (D), separated into primary, secondary, and tertiary branches, of neurons shown in (B). (E) Quantification of dendrite numbers separated into total apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites. (F) Epac2 knockdown in vivo induced a selective reduction in basal dendrite length. (G) Quantification of dendrite branch numbers separated into basal/apical, primary/secondary/tertiary order branches. Epac2 knockdown in vivo induced a loss of basal dendrites, specifically driven by a loss of secondary and tertiary basal dendrites. (H) Epac2 knockdown in vivo selectively reduced secondary and tertiary basal dendrite lengths. *p<0.05, ***p<0.001; scale bars, 100 µm (A); 50 µm (B–D).
Mentions: Epac2, an upstream regulator of Rap activity, is involved in regulating synapse morphology [22], but its role in regulating the architecture of the dendritic tree in cortical neurons is unknown. We first tested whether altered expression of Epac2 affected the maintenance of dendritic arbors in vivo, using in utero electroporation (IUEP) [30] to knock down protein expression (Figure S1A). Using a previously characterized RNA interference (RNAi) construct selective for Epac2 [22], we coinjected either Epac2-RNAi, or control (pGSuper), with pCAG-eGFP into the subventricular zone of E16.5 mouse embryos to specifically target layer 2/3 neurons. Electroporated constructs were allowed to express until brain harvesting and sectioning on postnatal day 28 (P28). In vivo knockdown of the Epac2 protein via Epac2-RNAi was confirmed at P28 by immunohistochemistry of cortical sections. Quantification of GFP-positive cells revealed that Epac2 was knocked down by ∼75% compared to GFP-negative cells (Figure S1B–C). Electroporated neurons were specifically found in layer 2/3 (Figure 1A). When we analyzed dendritic morphology by measuring apical and basal dendritic number and length in GFP-positive cells, we observed that long-term knockdown of Epac2 expression specifically reduced basal dendrites. Examination of basal arbors of layer 2/3 neurons from P28 mice expressing GFP and Epac2-RNAi (Figure 1B–F) revealed a ∼42% reduction in basal dendritic branches and a ∼54% reduction in basal dendritic length (p<0.001; Figure 1E–F) compared to cells from P28 mice expressing GFP and control construct. This effect on basal dendritic morphology was mediated by a ∼30% reduction in secondary and a ∼65% reduction in tertiary order basal branch number (p<0.05, 0.001; Figure 1G, Table S1A). Additionally, basal dendritic length was reduced by ∼24% in secondary branches and ∼55% in tertiary branches (p<0.05, 0.001; Figure 1H, Table S1B). The effect of Epac2 knockdown from E16.5 to P28 was specific for basal dendritic arbors, as Epac2-RNAi had no effect on apical dendrite branch number or length in these cells (Figure 1E–H, Table S1A–B).

Bottom Line: We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect.Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology.These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

Show MeSH
Related in: MedlinePlus