Tovar LM et al. (2023), Understanding the Role of ATP Release through C...

XB-ART-59723

Int J Mol Sci 2023 Jan 21;243:. doi: 10.3390/ijms24032159.

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Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Tovar LM , Burgos CF , Yévenes GE , Moraga-Cid G , Fuentealba J , Coddou C , Bascunan-Godoy L , Catrupay C , Torres A , Castro PA .

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Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

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Species referenced: Xenopus laevis
Genes referenced: fgf2 gja1 gja10 gja3 gja4 gja4.2 gja9 gjb1 gjb2 gjb3 gjb5 gjb7 gjc1 gjc2 gjd4 sub1
GO keywords: neural plate development [+]

Phenotypes: Xla Wt + carbenoxolone (Fig. 2 B) [+]

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	Figure 1. Cx46 and Cx32 transcripts are enriched during Xenopus laevis Neurulation. (A) RT-qPCR of Cx43, Cx46, Cx45, Cx32, Cx26, Cx31, and Cx25 in the process of formation of the neural tube of Xenopus laevis N = 4. (B) Comparative analysis of expression of Cxs in the closure of the neural tube of Xenopus laevis N = 4. Transcriptional expression was normalized to the sub-1 control gene. One-way ANOVA, Dunnett’s correction, (* = p < 0.05; = p < 0.01; * = p < 0.001; **** = p < 0.0001; ns = not significant). The error bars of the data correspond to the standard deviation.
	Figure 2. Pharmacological blockade of Cxs with CBX induces NTDs. (A) The diagram depicts the stage (Stg) where CBX was initially applied to embryos (Stg 12.5 = 14 hpf). (B) Representative photographs of embryos at stg 20 under control conditions (top) or treated with CBX (30 or 50 μM, middle and bottom, respectively). Dashed white lines mark the border between superficial neural and non-neural ectoderm, and dashed black lines indicate midline. Red arrowheads indicate the distance of neural folds (closed phenotype = normal; open phenotype = abnormal); scale bar = 500 μm. (C) The graph shows the percentage of open and closed phenotypes of embryos treated in the absence (control = ctl) or presence of CBX (3–300 µM; IC50 = 21.05 μM ± 1.22). (D) Quantification of open phenotype in embryos at stg 20 treated with CBX (IC50 = 58.56 μM ± 2.98). (E) The phenotype of tadpoles (stg 41–43) treated with CBX at stg 12.5–20. Scale bar = 1 mm. (F) The graph shows the effect of CBX on the length of Xenopus tadpoles. Independent fertilized embryos (N = 6) were analyzed for conditions (10 experimental replicates). One-way ANOVA, Dunnett’s correction, (* = p < 0.001; ** = p < 0.0001; ns = not significant). Error bars represent the standard deviation.
	Figure 3. Lucifer Yellow uptake is modulated by FGF2 and DTT in neural plates. (A–G) Representative images of the neural tube closure process in the control condition, DTT, DTT/CBX, DTT/ENX, FGF2, FGF2/CBX, and FGF2/ENX. (A’–G’) Neural plate cell fluorescence, illustrating LY uptake in control conditions, DTT, DTT/CBX, DTT/ENX, FGF2, FGF2/CBX, and FGF2/ENX. Scale bar: 200 µm. The upper panel images show a magnification of the boxes in (A’,B’,E’) signaling LY uptake, respectively. Scale bar (A’’,B’’,E’’): 20 μm. Scale bar magnified boxes: 20 μm. (H) Comparison of positive LY cells under redox potential (DTT) and FGF2 conditions, quantification normalized to control condition (MMR 10%). (I) Quantification of LY cells after exposure to DTT. (J) Quantification of LY cells after exposure to FGF2. N = 3 groups of independent fertilized embryos and 5 replicates for each condition. One-way ANOVA, Bonferroní comparison test, ( = p < 0.01; ** = p < 0.0001; ns = not significant). Error bars for data represent standard deviation.
	Figure 4. ATP Release during neurulation is regulated by HCs-Cxs modulators. Using luminescence assays (A), extracellular and intracellular ATP (nM) were quantified and normalized to the calibration curve. (B) Embryos treated with redox potentials. (C) Embryos treated with mitogenic factors. The results correspond to at least four experiments conducted independently (N = 4), for each condition. One-way ANOVA, Bonferroní comparison test, (* = p < 0.05; = p < 0.01; * = p < 0.001; **** = p < 0.0001; ns = not significant.). The error bars for the data represent the standard deviation.
	Figure 5. Cavities identified in the hemichannel of connexin 46. Hemichannel is visualized in a 90° rotation (x-axis). (A) Structure of HC-Cx46 representing the three best cavities; cavity 1 = 0.795, cavity 12 = 0.883, and cavity 17 = 0.634 calculation carried out with Fpocket. (B–D) The chain structure of Cx46 represents the three best cavities found by Fpocket. Summary table, detailing drugability score, amino acid residues, chains, and domains.
	Figure 6. Molecular docking of CBX and ENX in the structure of HCs-Cx46. (A–C) Representative interaction between HCs-Cx46, CBX (magenta), and ENX (light blue) in the three cavity predicted previously. All the chains are identical, each monomer is represented with a different color to facilitate the identification of the regions between subunits. Comparison of CBX/ENX docking in the NT domains of HCs-Cx46. 2D diagram of the interactions identified for HCs-Cx46. The CBX and ENX binding site residues that interact with the NT of HC-Cx46 are shown schematically and colored according to their physicochemical properties. The arrows indicate the directionality of the hydrogen bond and the degraded line represent a salt bridge. Plots of docking scores and theoretical binding ΔG between CBX/ENX interactions with HCs-Cx46.
	Figure S1. RT-PCR for Cx43, Cx46, Cx45, Cx32, Cx26, Cx31 y Cx25 at different stages of the development of Xenopus laevis. Lane 1. Gas (Gastrula stage 10), Lane 2. Ely (early stage 12.5), Lane 3. Int (Intermediate stage 14), Lane 4. Lte (Late stage 19-20), Lane 5. A.B. (Adult Brain) and Lane 6. Mcr (Marker 100 bp).
	Figure S2. Pharmacological blockade of Cxs with ENX induces NTDs. A. The diagram depicts the stage (Stg) where ENX was initially applied to embryos (Stg 12.5 = 14 hpf). B. Representative photographs of embryos at stg 20 under control conditions (top) or treated with ENX (30 or 50 M, middle and bottom, respectively). Dashed white lines mark the border between superficial neural and non-neural ectoderm, dashed black lines indicate midline. Red arrow heads indicate the distance of neural folds (closed phenotype= normal; open phenotype= abnormal); scale bar = 500 μm C. The graph shows the percentage of open and closed phenotype of embryos treated in the absence (control= ctl) or presence of ENX (3-300 µM; IC50= 47.83 μM ± 2.31) D. Quantification of open phenotype in embryos at stg 20 treated with ENX (IC50= 67.74 μM ± 4.19). E. Phenotype of tadpoles (stg 41-43) treated with ENX at stg 12.5-20. Scale bar = 1 mm. F. The graph shows the effect of CBX on the length of Xenopus tadpoles. Independent fertilized embryos (N=6) were analyzed for condition (10 experimental replicates). One-way ANOVA, Dunnett's correction, = p<0.01; * = p<0.001; ns= not significant). Error bars represent the standard deviation.

	Figure S4. Cx46 is expressed during Neurulation in Xenopus laevis. A, Representative western blot showing the expression of cx46 in different stages of Neurulation in Xenopus. Lane 1 corresponds to stage 12; lanes 2 corresponds to stage 15; lane 3 corresponds to stage 20. A molecular weight marker is indicated with an arrow. B. Densitometric quantification of cx46 signal normalized by the total amount of protein loaded in each lane. Bars correspond to average ± ES. (** = p<0.01; n=4). C. Dot Blot Assay Anti-Cx46. a-c. Positive control (peptide sequence: CRLPSRNSRHSSNRS). d. Mouse cerebral Cortex. e. Xenopus laevis embryos stage 20.

References [+] :

Abbracchio, Purinergic signalling in the nervous system: an overview. 2009, Pubmed

	Figure 1. Cx46 and Cx32 transcripts are enriched during Xenopus laevis Neurulation. (A) RT-qPCR of Cx43, Cx46, Cx45, Cx32, Cx26, Cx31, and Cx25 in the process of formation of the neural tube of Xenopus laevis N = 4. (B) Comparative analysis of expression of Cxs in the closure of the neural tube of Xenopus laevis N = 4. Transcriptional expression was normalized to the sub-1 control gene. One-way ANOVA, Dunnett’s correction, (* = p < 0.05; = p < 0.01; * = p < 0.001; **** = p < 0.0001; ns = not significant). The error bars of the data correspond to the standard deviation.
	Figure 2. Pharmacological blockade of Cxs with CBX induces NTDs. (A) The diagram depicts the stage (Stg) where CBX was initially applied to embryos (Stg 12.5 = 14 hpf). (B) Representative photographs of embryos at stg 20 under control conditions (top) or treated with CBX (30 or 50 μM, middle and bottom, respectively). Dashed white lines mark the border between superficial neural and non-neural ectoderm, and dashed black lines indicate midline. Red arrowheads indicate the distance of neural folds (closed phenotype = normal; open phenotype = abnormal); scale bar = 500 μm. (C) The graph shows the percentage of open and closed phenotypes of embryos treated in the absence (control = ctl) or presence of CBX (3–300 µM; IC50 = 21.05 μM ± 1.22). (D) Quantification of open phenotype in embryos at stg 20 treated with CBX (IC50 = 58.56 μM ± 2.98). (E) The phenotype of tadpoles (stg 41–43) treated with CBX at stg 12.5–20. Scale bar = 1 mm. (F) The graph shows the effect of CBX on the length of Xenopus tadpoles. Independent fertilized embryos (N = 6) were analyzed for conditions (10 experimental replicates). One-way ANOVA, Dunnett’s correction, (* = p < 0.001; ** = p < 0.0001; ns = not significant). Error bars represent the standard deviation.
	Figure 3. Lucifer Yellow uptake is modulated by FGF2 and DTT in neural plates. (A–G) Representative images of the neural tube closure process in the control condition, DTT, DTT/CBX, DTT/ENX, FGF2, FGF2/CBX, and FGF2/ENX. (A’–G’) Neural plate cell fluorescence, illustrating LY uptake in control conditions, DTT, DTT/CBX, DTT/ENX, FGF2, FGF2/CBX, and FGF2/ENX. Scale bar: 200 µm. The upper panel images show a magnification of the boxes in (A’,B’,E’) signaling LY uptake, respectively. Scale bar (A’’,B’’,E’’): 20 μm. Scale bar magnified boxes: 20 μm. (H) Comparison of positive LY cells under redox potential (DTT) and FGF2 conditions, quantification normalized to control condition (MMR 10%). (I) Quantification of LY cells after exposure to DTT. (J) Quantification of LY cells after exposure to FGF2. N = 3 groups of independent fertilized embryos and 5 replicates for each condition. One-way ANOVA, Bonferroní comparison test, ( = p < 0.01; ** = p < 0.0001; ns = not significant). Error bars for data represent standard deviation.
	Figure 4. ATP Release during neurulation is regulated by HCs-Cxs modulators. Using luminescence assays (A), extracellular and intracellular ATP (nM) were quantified and normalized to the calibration curve. (B) Embryos treated with redox potentials. (C) Embryos treated with mitogenic factors. The results correspond to at least four experiments conducted independently (N = 4), for each condition. One-way ANOVA, Bonferroní comparison test, (* = p < 0.05; = p < 0.01; * = p < 0.001; **** = p < 0.0001; ns = not significant.). The error bars for the data represent the standard deviation.
	Figure 5. Cavities identified in the hemichannel of connexin 46. Hemichannel is visualized in a 90° rotation (x-axis). (A) Structure of HC-Cx46 representing the three best cavities; cavity 1 = 0.795, cavity 12 = 0.883, and cavity 17 = 0.634 calculation carried out with Fpocket. (B–D) The chain structure of Cx46 represents the three best cavities found by Fpocket. Summary table, detailing drugability score, amino acid residues, chains, and domains.
	Figure 6. Molecular docking of CBX and ENX in the structure of HCs-Cx46. (A–C) Representative interaction between HCs-Cx46, CBX (magenta), and ENX (light blue) in the three cavity predicted previously. All the chains are identical, each monomer is represented with a different color to facilitate the identification of the regions between subunits. Comparison of CBX/ENX docking in the NT domains of HCs-Cx46. 2D diagram of the interactions identified for HCs-Cx46. The CBX and ENX binding site residues that interact with the NT of HC-Cx46 are shown schematically and colored according to their physicochemical properties. The arrows indicate the directionality of the hydrogen bond and the degraded line represent a salt bridge. Plots of docking scores and theoretical binding ΔG between CBX/ENX interactions with HCs-Cx46.
	Figure S1. RT-PCR for Cx43, Cx46, Cx45, Cx32, Cx26, Cx31 y Cx25 at different stages of the development of Xenopus laevis. Lane 1. Gas (Gastrula stage 10), Lane 2. Ely (early stage 12.5), Lane 3. Int (Intermediate stage 14), Lane 4. Lte (Late stage 19-20), Lane 5. A.B. (Adult Brain) and Lane 6. Mcr (Marker 100 bp).
	Figure S2. Pharmacological blockade of Cxs with ENX induces NTDs. A. The diagram depicts the stage (Stg) where ENX was initially applied to embryos (Stg 12.5 = 14 hpf). B. Representative photographs of embryos at stg 20 under control conditions (top) or treated with ENX (30 or 50 M, middle and bottom, respectively). Dashed white lines mark the border between superficial neural and non-neural ectoderm, dashed black lines indicate midline. Red arrow heads indicate the distance of neural folds (closed phenotype= normal; open phenotype= abnormal); scale bar = 500 μm C. The graph shows the percentage of open and closed phenotype of embryos treated in the absence (control= ctl) or presence of ENX (3-300 µM; IC50= 47.83 μM ± 2.31) D. Quantification of open phenotype in embryos at stg 20 treated with ENX (IC50= 67.74 μM ± 4.19). E. Phenotype of tadpoles (stg 41-43) treated with ENX at stg 12.5-20. Scale bar = 1 mm. F. The graph shows the effect of CBX on the length of Xenopus tadpoles. Independent fertilized embryos (N=6) were analyzed for condition (10 experimental replicates). One-way ANOVA, Dunnett's correction, = p<0.01; * = p<0.001; ns= not significant). Error bars represent the standard deviation.

	Figure S4. Cx46 is expressed during Neurulation in Xenopus laevis. A, Representative western blot showing the expression of cx46 in different stages of Neurulation in Xenopus. Lane 1 corresponds to stage 12; lanes 2 corresponds to stage 15; lane 3 corresponds to stage 20. A molecular weight marker is indicated with an arrow. B. Densitometric quantification of cx46 signal normalized by the total amount of protein loaded in each lane. Bars correspond to average ± ES. (** = p<0.01; n=4). C. Dot Blot Assay Anti-Cx46. a-c. Positive control (peptide sequence: CRLPSRNSRHSSNRS). d. Mouse cerebral Cortex. e. Xenopus laevis embryos stage 20.