J Mol Neurosci DOI 10.1007/s12031-015-0515-8
Pcdh11x Negatively Regulates Dendritic Branching Cuiying Wu & Lijun Niu & Zhongjie Yan & Chong Wang & Ning Liu & Yiwu Dai & Peng Zhang & Ruxiang Xu
Received: 9 December 2014 / Accepted: 4 February 2015 # Springer Science+Business Media New York 2015
Abstract Proper formation of neuronal dendritic branching is crucial for correct brain function. The number and distribution of receptive synaptic contacts are defined by the size and shape of dendritic arbors. Our previous research found that protocadherin 11 X-linked protein (Pcdh11x) is predominantly expressed in neurons and has an influence on dendritic branching. In this study, gain-of-function and loss-offunction experiments revealed that Pcdh11x acts as a negative regulator of dendritic branching in cultured cortical neurons derived from embryonic day 16 mice. Overexpression of wild-type Pcdh11x (Pcdh11x-GFP) reduced dendritic complexity, whereas knockdown of Pcdh11x increased dendritic branching. It was further demonstrated that Pcdh11x activates PI3K/AKT signaling to negatively regulate dendritic branching. Keywords Protocadherin 11 X-linked protein . Pcdh11x . Dendritic branching . Phosphoinositide-3-kinase (PI3K)/AKT
Introduction The chemoaffinity hypothesis states that the expression of specific combinations of diverse transmembrane tagging proteins is required for precise neural circuitry (Hattori et al. C. Wu : L. Niu : C. Wang : N. Liu : Y. Dai : P. Zhang (*) : R. Xu (*) Affiliated Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing, China e-mail: [email protected] e-mail: [email protected] Z. Yan Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, China
2008; Sperry 1963; Zipursky and Sanes 2010). During the development and maintenance of neural circuits, the formation of elaborate dendritic branches and spine patterns is critical (Jan and Jan 2010). Dendrite growth is a dynamic process, and dendrites continue to extend and branch profusely long after migration is complete, significantly contributing to postnatal brain growth. Dendritic morphology is closely related to the correct wiring of neuronal circuits and, as a result, their function (Gidon and Segev 2012; Lavzin et al. 2012; Branco and Hausser 2011; Branco et al. 2010; Parrish et al. 2007; Hausser et al. 2000). Dendrites extend away from the soma into the target field depending on guidance cues and are able to branch multiple times, with secondary and tertiary branching. Despite having an essential role in brain function, much less is known about dendrite growth compared with other fields of nervous system development such as axon guidance. Proteins from the cadherin superfamily and their signaling pathways act as diverse regulators of dendrite growth and spine morphogenesis (Lin and Koleske 2010; Takeichi 2007; Ye and Jan 2005; Yu and Malenka 2003). The cadherins are a class of transmembrane proteins, which includes classic cadherins, desmosomal cadherins, protocadherins (Pcdhs), and other proteins (Morishita and Yagi 2007). Pcdhs are classified into two subgroups, clustered and non-clustered Pcdhs, based on genomic structure (Morishita and Yagi 2007). Clustered Pcdhs play important roles in dendritic development and spine formation (Suo et al. 2012). For example, Pcdhα and Pcdhγ clusters are required for dendrite and spine morphogenesis because Pcdhα or Pcdhγ cluster knockdown mice display neural dendritic defects (Suo et al. 2012). Furthermore, methylation of clustered Pcdh promoters by the de novo DNA methyltransferase Dnmt3b during early embryogenesis may promote dendritic arborization in Purkinje cells (Toyoda et al. 2014). Our previous results indicate that Pcdh11x may suppress neuronal differentiation
J Mol Neurosci
(Zhang et al. 2014), and it was also found that Pcdh11x affects neuronal dendrite branching. The present study was primarily focused on the effects of Pcdh11x on dendritic branching. Using cultured cortical neurons as a cellular model, Pcdh11x suppressed dendrite branching. The suppression of dendritic growth by Pcdh11x is consistent with the reduction in its expression during dendrite maturation. Further experiments were conducted to explore the possible molecular mechanisms that underlie the effects of Pcdh11x on neuronal dendrite formation.
role of Pcdh11x in dendritic branching, dissociated neurons were electrotransfected with DNA encoding Pcdh11x-shRNA and pCAG-Pcdh11x-GFP (4D-Nucleofector™ System, Lonza). Transfected neurons were then plated at a density of 5×105 cells onto 6- or 24-well plates coated with poly-Llysine and laminin. After culturing for 24 h, the medium was changed to neurobasal medium supplemented with 2 % B27 and 0.5 mM GlutaMAX-I. At 3 or 6 days in vitro (DIV), neurons were fixed with 4 % paraformaldehyde, and dendritic branching was analyzed. Neuronal transfection was performed according to the manufacturer’s instructions.
Methods
RNA Interference
Plasmids, Reagents, and Antibodies
The sequence of the Pcdh11x small interfering RNA (siRNA) was as follows: 5′-GCACCGUUAUUCCCAACAATT-3′ (Zhang et al. 2014). A control siRNA was used with the following sequence 5′-UUCUCCGAACGUGUCACGUTT-3′, which is unable to knockdown the expression of any known proteins. Here, the short hairpin RNA (shRNA) system replaced the siRNA system with the same sequences.
Dulbecco’s modified Eagle’s medium (DMEM/F12 1:1 media, HyClone, SH3002301B); fetal bovine serum (FBS, Gibco, 10099141); poly-L-lysine hydrobromide (SigmaAldrich, Saint Louis, MO, P7890); laminin from EngelbrethHolm-Swarm murine sarcoma basement membrane (SigmaAldrich, Saint Louis, MO, L2020); neurobasal medium (Invitrogen, Carlsbad, CA, 21103-049); B-27® serum-free supplement (Invitrogen, Carlsbad, CA, 12587010, 2 %); N2 supplement (Invitrogen, Carlsbad, CA, 17502-048, 1 %); GlutaMAX-I (Invitrogen, Carlsbad, CA, 35050-061); DNase I (Roche, LOT 10932100); paraformaldehyde (SigmaAldrich, Saint Louis, MO, 158127); protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, EDTA-Free Thermo, 87785); lysis buffer (ProteoJET™ Mammalian Cell Lysis Reagent, Fermentas, K0301); bicinchoninic acid (BCA) assay kit (Pierce, 23227); 5× loading buffer (Beyotime, P0015); polyvinylidene difluoride (PVDF) membranes (Millipore, IPFL00010); bovine serum albumin (BSA, Sigma, A2153); Pcdh11x antibody (E-13, Santa Cruz, SC-103726); AKT antibody (Cell signaling, 9272); AKT Ser473 antibody (Cell signaling, phospho-Akt Ser473 (587 F11) mouse mAb, 4051); phosphoinositide-3-kinase (PI3K) antibody (Cell signaling, 4255); β-actin antibody (Cell signaling, 3700); electrochemilluminesence (ECL) solutions (Pierce, 34095); PI3K inhibitor (LY294002) (Sigma-Aldrich, L9908); ankyrin G (Abcam, Cambridge, MA, ab104896); mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector lab, H-1200); secondary antibody (Invitrogen, Carlsbad, CA, Alexa Fluor® 594 rabbit anti-rabbit IgG (H + L), A-11012). Neuronal Culture and Transfection Cortical explants isolated from day 16 mouse embryos of either sex were digested with 0.25 % trypsin for 5 min at 37 °C, followed by gentle pipetting with DNase I (20 U, 50 μl) in medium (DMEM/F12 with 10 % FBS). To test the
Western Blotting Analysis Cell cultures were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer containing protease inhibitor cocktail (1:100). The cell lysates were centrifuged at 17,000×g for 20 min at 4 °C to yield the total protein extract in the supernatant. Protein concentrations in the cell supernatants were measured with a BCA assay. Equal amounts of samples (50 μg) were denatured with 5× loading buffer and subjected to 10 or 6 % SDS-PAGE. After separation, proteins were transferred to PVDF membranes. The membranes were blocked with 5 % non-fat milk in TBST (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, and 0.05 % Tween 20) for 1 h at room temperature and incubated with primary antibodies (Pcdh11x, AKT, AKT Ser473, PI3K, β-actin) overnight at 4 °C. After washing with TBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, washed again, and then developed with ECL solution. The immunoreactive bands were scanned and analyzed quantitatively by densitometry with ImageJ (http://rsb.info.nih.gov/ij/). Tissue Preparation and Immunofluorescence After transfection, the neurons were fixed with paraformaldehyde (4 %) in 0.1 M PBS for 30 min and then washed three times, each time for 5 min, in 0.01 M PBS at room temperature. The neurons were blocked with 10 % BSA and 0.1 % Triton X-100 at 37 °C for 1 h. Neuronal staining was performed with ankyrin G diluted in 0.1 M PBS with 10 %
J Mol Neurosci
BSA overnight at 4 °C. On the following day, sections were washed three times, each time for 5 min, in 0.01 M PBS at room temperature and then incubated in secondary antibody for 1 h at 37 °C. The stained cells were mounted using mounting medium with DAPI.
±standard error (SEM). Comparisons between two groups were made using the Student’s t test. Comparisons among three or more groups were made using one-way analysis of variance, followed by Newman–Keuls tests. Data marked with asterisks are significantly different from the control as follows: ***P
Pcdh11x Negatively Regulates Dendritic Branching Cuiying Wu & Lijun Niu & Zhongjie Yan & Chong Wang & Ning Liu & Yiwu Dai & Peng Zhang & Ruxiang Xu
Received: 9 December 2014 / Accepted: 4 February 2015 # Springer Science+Business Media New York 2015
Abstract Proper formation of neuronal dendritic branching is crucial for correct brain function. The number and distribution of receptive synaptic contacts are defined by the size and shape of dendritic arbors. Our previous research found that protocadherin 11 X-linked protein (Pcdh11x) is predominantly expressed in neurons and has an influence on dendritic branching. In this study, gain-of-function and loss-offunction experiments revealed that Pcdh11x acts as a negative regulator of dendritic branching in cultured cortical neurons derived from embryonic day 16 mice. Overexpression of wild-type Pcdh11x (Pcdh11x-GFP) reduced dendritic complexity, whereas knockdown of Pcdh11x increased dendritic branching. It was further demonstrated that Pcdh11x activates PI3K/AKT signaling to negatively regulate dendritic branching. Keywords Protocadherin 11 X-linked protein . Pcdh11x . Dendritic branching . Phosphoinositide-3-kinase (PI3K)/AKT
Introduction The chemoaffinity hypothesis states that the expression of specific combinations of diverse transmembrane tagging proteins is required for precise neural circuitry (Hattori et al. C. Wu : L. Niu : C. Wang : N. Liu : Y. Dai : P. Zhang (*) : R. Xu (*) Affiliated Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing, China e-mail: [email protected] e-mail: [email protected] Z. Yan Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, China
2008; Sperry 1963; Zipursky and Sanes 2010). During the development and maintenance of neural circuits, the formation of elaborate dendritic branches and spine patterns is critical (Jan and Jan 2010). Dendrite growth is a dynamic process, and dendrites continue to extend and branch profusely long after migration is complete, significantly contributing to postnatal brain growth. Dendritic morphology is closely related to the correct wiring of neuronal circuits and, as a result, their function (Gidon and Segev 2012; Lavzin et al. 2012; Branco and Hausser 2011; Branco et al. 2010; Parrish et al. 2007; Hausser et al. 2000). Dendrites extend away from the soma into the target field depending on guidance cues and are able to branch multiple times, with secondary and tertiary branching. Despite having an essential role in brain function, much less is known about dendrite growth compared with other fields of nervous system development such as axon guidance. Proteins from the cadherin superfamily and their signaling pathways act as diverse regulators of dendrite growth and spine morphogenesis (Lin and Koleske 2010; Takeichi 2007; Ye and Jan 2005; Yu and Malenka 2003). The cadherins are a class of transmembrane proteins, which includes classic cadherins, desmosomal cadherins, protocadherins (Pcdhs), and other proteins (Morishita and Yagi 2007). Pcdhs are classified into two subgroups, clustered and non-clustered Pcdhs, based on genomic structure (Morishita and Yagi 2007). Clustered Pcdhs play important roles in dendritic development and spine formation (Suo et al. 2012). For example, Pcdhα and Pcdhγ clusters are required for dendrite and spine morphogenesis because Pcdhα or Pcdhγ cluster knockdown mice display neural dendritic defects (Suo et al. 2012). Furthermore, methylation of clustered Pcdh promoters by the de novo DNA methyltransferase Dnmt3b during early embryogenesis may promote dendritic arborization in Purkinje cells (Toyoda et al. 2014). Our previous results indicate that Pcdh11x may suppress neuronal differentiation
J Mol Neurosci
(Zhang et al. 2014), and it was also found that Pcdh11x affects neuronal dendrite branching. The present study was primarily focused on the effects of Pcdh11x on dendritic branching. Using cultured cortical neurons as a cellular model, Pcdh11x suppressed dendrite branching. The suppression of dendritic growth by Pcdh11x is consistent with the reduction in its expression during dendrite maturation. Further experiments were conducted to explore the possible molecular mechanisms that underlie the effects of Pcdh11x on neuronal dendrite formation.
role of Pcdh11x in dendritic branching, dissociated neurons were electrotransfected with DNA encoding Pcdh11x-shRNA and pCAG-Pcdh11x-GFP (4D-Nucleofector™ System, Lonza). Transfected neurons were then plated at a density of 5×105 cells onto 6- or 24-well plates coated with poly-Llysine and laminin. After culturing for 24 h, the medium was changed to neurobasal medium supplemented with 2 % B27 and 0.5 mM GlutaMAX-I. At 3 or 6 days in vitro (DIV), neurons were fixed with 4 % paraformaldehyde, and dendritic branching was analyzed. Neuronal transfection was performed according to the manufacturer’s instructions.
Methods
RNA Interference
Plasmids, Reagents, and Antibodies
The sequence of the Pcdh11x small interfering RNA (siRNA) was as follows: 5′-GCACCGUUAUUCCCAACAATT-3′ (Zhang et al. 2014). A control siRNA was used with the following sequence 5′-UUCUCCGAACGUGUCACGUTT-3′, which is unable to knockdown the expression of any known proteins. Here, the short hairpin RNA (shRNA) system replaced the siRNA system with the same sequences.
Dulbecco’s modified Eagle’s medium (DMEM/F12 1:1 media, HyClone, SH3002301B); fetal bovine serum (FBS, Gibco, 10099141); poly-L-lysine hydrobromide (SigmaAldrich, Saint Louis, MO, P7890); laminin from EngelbrethHolm-Swarm murine sarcoma basement membrane (SigmaAldrich, Saint Louis, MO, L2020); neurobasal medium (Invitrogen, Carlsbad, CA, 21103-049); B-27® serum-free supplement (Invitrogen, Carlsbad, CA, 12587010, 2 %); N2 supplement (Invitrogen, Carlsbad, CA, 17502-048, 1 %); GlutaMAX-I (Invitrogen, Carlsbad, CA, 35050-061); DNase I (Roche, LOT 10932100); paraformaldehyde (SigmaAldrich, Saint Louis, MO, 158127); protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, EDTA-Free Thermo, 87785); lysis buffer (ProteoJET™ Mammalian Cell Lysis Reagent, Fermentas, K0301); bicinchoninic acid (BCA) assay kit (Pierce, 23227); 5× loading buffer (Beyotime, P0015); polyvinylidene difluoride (PVDF) membranes (Millipore, IPFL00010); bovine serum albumin (BSA, Sigma, A2153); Pcdh11x antibody (E-13, Santa Cruz, SC-103726); AKT antibody (Cell signaling, 9272); AKT Ser473 antibody (Cell signaling, phospho-Akt Ser473 (587 F11) mouse mAb, 4051); phosphoinositide-3-kinase (PI3K) antibody (Cell signaling, 4255); β-actin antibody (Cell signaling, 3700); electrochemilluminesence (ECL) solutions (Pierce, 34095); PI3K inhibitor (LY294002) (Sigma-Aldrich, L9908); ankyrin G (Abcam, Cambridge, MA, ab104896); mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector lab, H-1200); secondary antibody (Invitrogen, Carlsbad, CA, Alexa Fluor® 594 rabbit anti-rabbit IgG (H + L), A-11012). Neuronal Culture and Transfection Cortical explants isolated from day 16 mouse embryos of either sex were digested with 0.25 % trypsin for 5 min at 37 °C, followed by gentle pipetting with DNase I (20 U, 50 μl) in medium (DMEM/F12 with 10 % FBS). To test the
Western Blotting Analysis Cell cultures were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer containing protease inhibitor cocktail (1:100). The cell lysates were centrifuged at 17,000×g for 20 min at 4 °C to yield the total protein extract in the supernatant. Protein concentrations in the cell supernatants were measured with a BCA assay. Equal amounts of samples (50 μg) were denatured with 5× loading buffer and subjected to 10 or 6 % SDS-PAGE. After separation, proteins were transferred to PVDF membranes. The membranes were blocked with 5 % non-fat milk in TBST (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, and 0.05 % Tween 20) for 1 h at room temperature and incubated with primary antibodies (Pcdh11x, AKT, AKT Ser473, PI3K, β-actin) overnight at 4 °C. After washing with TBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, washed again, and then developed with ECL solution. The immunoreactive bands were scanned and analyzed quantitatively by densitometry with ImageJ (http://rsb.info.nih.gov/ij/). Tissue Preparation and Immunofluorescence After transfection, the neurons were fixed with paraformaldehyde (4 %) in 0.1 M PBS for 30 min and then washed three times, each time for 5 min, in 0.01 M PBS at room temperature. The neurons were blocked with 10 % BSA and 0.1 % Triton X-100 at 37 °C for 1 h. Neuronal staining was performed with ankyrin G diluted in 0.1 M PBS with 10 %
J Mol Neurosci
BSA overnight at 4 °C. On the following day, sections were washed three times, each time for 5 min, in 0.01 M PBS at room temperature and then incubated in secondary antibody for 1 h at 37 °C. The stained cells were mounted using mounting medium with DAPI.
±standard error (SEM). Comparisons between two groups were made using the Student’s t test. Comparisons among three or more groups were made using one-way analysis of variance, followed by Newman–Keuls tests. Data marked with asterisks are significantly different from the control as follows: ***P
