RESEARCH ARTICLE
Astrocyte Transforming Growth Factor Beta 1 Promotes Inhibitory Synapse Formation via CaM Kinase II Signaling Luan Pereira Diniz,1 Vanessa Tortelli,1 Matheus Nunes Garcia,1 Ana Paula B ergamo Ara ujo,1 Helen M. Melo,2 Gisele S. Seixas da Silva,2 Fernanda G. De Felice,2 Soniza Vieira Alves-Leon,3 Jorge Marcondes de Souza,3 Luciana Ferreira Rom~ ao,4 Newton Gonc¸alves Castro,1 and Fl avia Carvalho Alcantara Gomes1 The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-b1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-b pathway. TGF-b1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-b1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-b1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-b1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. GLIA 2014;00:000–000
Key words: inhibitory synapse, astrocyte, TGF-b1, CaMKII, Neuroligin 2
Introduction
I
dentification of cellular and molecular events underlying synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for glial cells, mainly astrocytes, in neuronal differentiation (Martinez and Gomes, 2002; Spohr et al., 2008, 2011) and circuitry formation has emerged within the last
decade (Araque et al., 1999; Mazzanti and Haydon, 2003; Schafer et al., 2013; Verbich et al., 2012). Astrocytes regulate excitatory synapse formation through contact molecules (Hama et al., 2004) and soluble factors, such as cholesterol (Mauch et al., 2001), thrombospondin (Christopherson et al., 2005), hevin (Kucukdereli et al., 2011), glyplican (Allen et al., 2012), and TGF-b1 (Diniz et al., 2012).
View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22713 Published online Month 00, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Feb 21, 2014, Accepted for publication June 18, 2014. Address correspondence to Fl avia Carvalho Alcantara Gomes, Instituto de Ci^ encias Biom edicas, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brasil. E-mail: [email protected] encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2Instituto de Bioquımica M edica, Universidade FedFrom the 1Instituto de Ci^ ario Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; eral do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 3Hospital Universit 4 UFRJ/P olo Maca e, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. Vanessa Tortelli and Matheus Nunes Garcia have equally contributed to the article. Additional Supporting Information may be found in the online version of this article.
C 2014 Wiley Periodicals, Inc. 1 V
Recent evidence indicated that, in addition to regulating excitatory synaptogenesis, astrocytes also affect inhibitory synapse formation (Eroglu and Barres, 2010). This event might involve distinct mechanisms such as glutamate release in response to activation of GABAB receptors (Kang et al., 1998), increase in Cl2 currents induced by GABA, contributing to the maintenance of GABAA receptors in the membrane (Liu et al., 1996, 1997). Studies have proposed that astrocytes induce inhibitory synapse formation through a soluble factor that promotes axonal outgrowth and neurotrophin signaling (Elmariah et al., 2005; Hughes et al., 2010). However, the molecule(s) and the molecular mechanisms that mediate astrocyte action in inhibitory synaptic transmission in the cerebral cortex remain unknown. Here, we investigated TGF-b1 as a possible glia-derived molecule involved in inhibitory synaptic formation in the cerebral cortex. The TGF-b superfamily is constituted by multifunctional polypeptide members, which perform essential functions in nervous system development and repair (Gomes et al., 2005; Krieglstein et al., 2011). TGF-b signaling pathway is critical for modulation of GABAA synaptic transmission and dendritic homeostasis. Expression of dominantnegative Smad mutants in the developing basal ganglia of mice impairs migration of GABAergic neurons (Maira et al., 2010). In addition, the hippocampus of transgenic Smad4deficient mouse shows stronger paired-pulse depression of GABAA currents (Sun et al., 2010). Also, the cerebella of these mice exhibit decreased numbers of Purkinje cells and parvalbumin-positive interneurons (Zhou et al., 2003). Despite this indirect evidence for a possible role of TGF-b in inhibitory synapse formation, the actual role of this factor in this event remains unknown. We recently showed that cortical astrocytes are important sources of TGF-b1 that has great impact on the excitatory synapse formation through a mechanism involving NMDA receptor activity (Diniz et al., 2012). Within this perspective we investigated whether TGF-b secreted by astrocytes could induce inhibitory synapse formation and the molecular mechanisms underlying this event. Here, we show that human and murine astrocytes regulate inhibitory synapse formation by TGF-b pathway in a mechanism involving NMDA receptor/CaMKII signaling and the transynaptic adhesion protein, Neuroligin 2. This work contributes to the understanding of the GABAergic synapse formation and sheds light on the mechanism underlying glial control of neural circuits during development and pathology.
Materials and Methods Animals Approximately 60 embryonic (E14; 14-day embryonic), 20 newborn (P0) and 16 three-month-old male Swiss mice were used. All
2
animal-use protocols were approved by the Animal Use Ethics Committee of the Federal University of Rio de Janeiro.
Murine Astrocyte Cultures Primary astrocyte cultures were prepared from neonatal Swiss mice (1 to 2 days old) cerebral cortex as previously described (Diniz et al., 2012; Gomes et al., 1999a,b). Briefly, the mice were decapitated, the cortices were isolated, and meninges carefully removed. Tissues were washed in phosphate-buffered saline (PBS) with 0.6% glucose (Sigma Chemical Co., St. Louis, MO) and dissociated into single cells in a medium consisting of Dulbecco’s minimum essential medium (DMEM) and nutrient mixture F12 (DMEM/F12, Invitrogen, Carlsbad, CA), enriched with glucose (3.3 3 1022 M), glutamine (2 3 1023 M) and sodium bicarbonate (0.3 3 1022 M). Dissociated cells were plated onto glass coverslips previously coated with poly-L-lysine (Sigma Chemical Co., St. Louis, MO), on a 24well plate (Corning Incorporated, Corning, NY), in DMEM/F12 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The cultures were incubated at 37 C in a humidified 5% CO2, 95% air chamber for 7 to 10 days until reaching confluence. After growing to confluence, cells were subjected to passages to generate pure astrocytic cultures. This protocol yields an astrocyteenriched culture constituted by more than 95% of GFAP (glial fibrillary acidic protein)-positive cells.
Human Astrocyte Cultures Adult primary human astrocytes were isolated from surgically resected anterior temporal lobe tissue, from patients selected for surgical treatment of temporal-lobe epilepsy associated with hippocampus sclerosis (TLE-HS). The pathological tissue targeted in surgery for these cases is the gliotic hippocampus, and the anterior temporal lobe resection is used merely as a surgical pathway to the diseased area. As previously described (Diniz et al., 2012), only healthy cortical tissue was used to produce astrocyte cultures. All patients gave written consent to the study, and the procedures were in agreement with the Brazilian Ministry of Health Ethics Committee. Briefly, tissues were washed in DMEM medium, mechanically dissociated, chopped into small (
Astrocyte Transforming Growth Factor Beta 1 Promotes Inhibitory Synapse Formation via CaM Kinase II Signaling Luan Pereira Diniz,1 Vanessa Tortelli,1 Matheus Nunes Garcia,1 Ana Paula B ergamo Ara ujo,1 Helen M. Melo,2 Gisele S. Seixas da Silva,2 Fernanda G. De Felice,2 Soniza Vieira Alves-Leon,3 Jorge Marcondes de Souza,3 Luciana Ferreira Rom~ ao,4 Newton Gonc¸alves Castro,1 and Fl avia Carvalho Alcantara Gomes1 The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-b1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-b pathway. TGF-b1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-b1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-b1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-b1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. GLIA 2014;00:000–000
Key words: inhibitory synapse, astrocyte, TGF-b1, CaMKII, Neuroligin 2
Introduction
I
dentification of cellular and molecular events underlying synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for glial cells, mainly astrocytes, in neuronal differentiation (Martinez and Gomes, 2002; Spohr et al., 2008, 2011) and circuitry formation has emerged within the last
decade (Araque et al., 1999; Mazzanti and Haydon, 2003; Schafer et al., 2013; Verbich et al., 2012). Astrocytes regulate excitatory synapse formation through contact molecules (Hama et al., 2004) and soluble factors, such as cholesterol (Mauch et al., 2001), thrombospondin (Christopherson et al., 2005), hevin (Kucukdereli et al., 2011), glyplican (Allen et al., 2012), and TGF-b1 (Diniz et al., 2012).
View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22713 Published online Month 00, 2014 in Wiley Online Library (wileyonlinelibrary.com). Received Feb 21, 2014, Accepted for publication June 18, 2014. Address correspondence to Fl avia Carvalho Alcantara Gomes, Instituto de Ci^ encias Biom edicas, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brasil. E-mail: [email protected] encias Biom edicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2Instituto de Bioquımica M edica, Universidade FedFrom the 1Instituto de Ci^ ario Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; eral do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 3Hospital Universit 4 UFRJ/P olo Maca e, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. Vanessa Tortelli and Matheus Nunes Garcia have equally contributed to the article. Additional Supporting Information may be found in the online version of this article.
C 2014 Wiley Periodicals, Inc. 1 V
Recent evidence indicated that, in addition to regulating excitatory synaptogenesis, astrocytes also affect inhibitory synapse formation (Eroglu and Barres, 2010). This event might involve distinct mechanisms such as glutamate release in response to activation of GABAB receptors (Kang et al., 1998), increase in Cl2 currents induced by GABA, contributing to the maintenance of GABAA receptors in the membrane (Liu et al., 1996, 1997). Studies have proposed that astrocytes induce inhibitory synapse formation through a soluble factor that promotes axonal outgrowth and neurotrophin signaling (Elmariah et al., 2005; Hughes et al., 2010). However, the molecule(s) and the molecular mechanisms that mediate astrocyte action in inhibitory synaptic transmission in the cerebral cortex remain unknown. Here, we investigated TGF-b1 as a possible glia-derived molecule involved in inhibitory synaptic formation in the cerebral cortex. The TGF-b superfamily is constituted by multifunctional polypeptide members, which perform essential functions in nervous system development and repair (Gomes et al., 2005; Krieglstein et al., 2011). TGF-b signaling pathway is critical for modulation of GABAA synaptic transmission and dendritic homeostasis. Expression of dominantnegative Smad mutants in the developing basal ganglia of mice impairs migration of GABAergic neurons (Maira et al., 2010). In addition, the hippocampus of transgenic Smad4deficient mouse shows stronger paired-pulse depression of GABAA currents (Sun et al., 2010). Also, the cerebella of these mice exhibit decreased numbers of Purkinje cells and parvalbumin-positive interneurons (Zhou et al., 2003). Despite this indirect evidence for a possible role of TGF-b in inhibitory synapse formation, the actual role of this factor in this event remains unknown. We recently showed that cortical astrocytes are important sources of TGF-b1 that has great impact on the excitatory synapse formation through a mechanism involving NMDA receptor activity (Diniz et al., 2012). Within this perspective we investigated whether TGF-b secreted by astrocytes could induce inhibitory synapse formation and the molecular mechanisms underlying this event. Here, we show that human and murine astrocytes regulate inhibitory synapse formation by TGF-b pathway in a mechanism involving NMDA receptor/CaMKII signaling and the transynaptic adhesion protein, Neuroligin 2. This work contributes to the understanding of the GABAergic synapse formation and sheds light on the mechanism underlying glial control of neural circuits during development and pathology.
Materials and Methods Animals Approximately 60 embryonic (E14; 14-day embryonic), 20 newborn (P0) and 16 three-month-old male Swiss mice were used. All
2
animal-use protocols were approved by the Animal Use Ethics Committee of the Federal University of Rio de Janeiro.
Murine Astrocyte Cultures Primary astrocyte cultures were prepared from neonatal Swiss mice (1 to 2 days old) cerebral cortex as previously described (Diniz et al., 2012; Gomes et al., 1999a,b). Briefly, the mice were decapitated, the cortices were isolated, and meninges carefully removed. Tissues were washed in phosphate-buffered saline (PBS) with 0.6% glucose (Sigma Chemical Co., St. Louis, MO) and dissociated into single cells in a medium consisting of Dulbecco’s minimum essential medium (DMEM) and nutrient mixture F12 (DMEM/F12, Invitrogen, Carlsbad, CA), enriched with glucose (3.3 3 1022 M), glutamine (2 3 1023 M) and sodium bicarbonate (0.3 3 1022 M). Dissociated cells were plated onto glass coverslips previously coated with poly-L-lysine (Sigma Chemical Co., St. Louis, MO), on a 24well plate (Corning Incorporated, Corning, NY), in DMEM/F12 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The cultures were incubated at 37 C in a humidified 5% CO2, 95% air chamber for 7 to 10 days until reaching confluence. After growing to confluence, cells were subjected to passages to generate pure astrocytic cultures. This protocol yields an astrocyteenriched culture constituted by more than 95% of GFAP (glial fibrillary acidic protein)-positive cells.
Human Astrocyte Cultures Adult primary human astrocytes were isolated from surgically resected anterior temporal lobe tissue, from patients selected for surgical treatment of temporal-lobe epilepsy associated with hippocampus sclerosis (TLE-HS). The pathological tissue targeted in surgery for these cases is the gliotic hippocampus, and the anterior temporal lobe resection is used merely as a surgical pathway to the diseased area. As previously described (Diniz et al., 2012), only healthy cortical tissue was used to produce astrocyte cultures. All patients gave written consent to the study, and the procedures were in agreement with the Brazilian Ministry of Health Ethics Committee. Briefly, tissues were washed in DMEM medium, mechanically dissociated, chopped into small (
