RESUMO
Leptin signaling within the nucleus of the solitary tract (NTS) contributes to the control of food intake, and injections of leptin into the NTS reduce meal size and increase the efficacy of vagus-mediated satiation signals. Leptin receptors (LepRs) are expressed by vagal afferents as well as by a population of NTS neurons. However, the electrophysiological properties of LepR-expressing NTS neurons have not been well characterized, and it is unclear how leptin might act on these neurons to reduce food intake. To address this question, we recorded from LepR-expressing neurons in horizontal brain slices containing the NTS from male and female LepR-Cre X Rosa-tdTomato mice. We found that the vast majority of NTS LepR neurons received monosynaptic innervation from vagal afferent fibers and LepR neurons exhibited large synaptic NMDA receptor (NMDAR)-mediated currents compared with non-LepR neurons. During high-frequency stimulation of vagal afferents, leptin increased the size of NMDAR-mediated currents, but not AMPAR-mediated currents. Leptin also increased the size of evoked EPSPs and the ability of low-intensity solitary tract stimulation to evoke action potentials in LepR neurons. These effects of leptin were blocked by bath applying a competitive NMDAR antagonist (DCPP-ene) or by an NMDAR channel blocker applied through the recording pipette (MK-801). Last, feeding studies using male rats demonstrate that intra-NTS injections of DCPP-ene attenuate reduction of overnight food intake following intra-NTS leptin injection. Our results suggest that leptin acts in the NTS to reduce food intake by increasing NMDAR-mediated currents, thus enhancing NTS sensitivity to vagal inputs.SIGNIFICANCE STATEMENT Leptin is a hormone that critically impacts food intake and energy homeostasis. The nucleus of the solitary tract (NTS) is activated by vagal afferents from the gastrointestinal tract, which promotes termination of a meal. Injection of leptin into the NTS inhibits food intake, while knockdown of leptin receptors (LepRs) in NTS neurons increases food intake. However, little was known about how leptin acts in the NTS neurons to inhibit food intake. We found that leptin increases the sensitivity of LepR-expressing neurons to vagal inputs by increasing NMDA receptor-mediated synaptic currents and that NTS NMDAR activation contributes to leptin-induced reduction of food intake. These findings suggest a novel mechanism by which leptin, acting in the NTS, could potentiate gastrointestinal satiation signals.
Assuntos
Potenciais Pós-Sinápticos Excitadores , Leptina/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Núcleo Solitário/metabolismo , Nervo Vago/metabolismo , Animais , Maleato de Dizocilpina/farmacologia , Ingestão de Alimentos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Prolina/análogos & derivados , Prolina/farmacologia , Piridinas/farmacologia , Ratos , Núcleo Solitário/citologia , Núcleo Solitário/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Nervo Vago/citologia , Nervo Vago/fisiologiaRESUMO
Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood. In this study, we reveal two novel targets of leptin in developing hippocampal neurons and address their role in synaptogenesis. First target is Kruppel-Like Factor 4 (KLF4), which we identified using a genome-wide target analysis strategy. We show that leptin upregulates KLF4 in hippocampal neurons and that leptin signaling is important for KLF4 expression in vivo. Furthermore, KLF4 is required for leptin-induced synaptogenesis, as shKLF4 blocks and upregulation of KLF4 phenocopies it. We go on to show that KLF4 requires its signal transducer and activator of transcription 3 (STAT3) binding site and thus potentially blocks STAT3 activity to induce synaptogenesis. Second, we show that leptin increases the expression of suppressor of cytokine signaling 3 (SOCS3), another well-known inhibitor of STAT3, in developing hippocampal neurons. SOCS3 is also required for leptin-induced synaptogenesis and sufficient to stimulate it alone. Finally, we show that constitutively active STAT3 blocks the effects of leptin on spine formation, while the targeted knockdown of STAT3 is sufficient to induce it. Overall, our data demonstrate that leptin increases the expression of both KLF4 and SOCS3, inhibiting the activity of STAT3 in the hippocampal neurons and resulting in the enhancement of glutamatergic synaptogenesis during neonatal development.
Assuntos
Hipocampo/efeitos dos fármacos , Leptina/farmacologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Feminino , Hipocampo/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Sinapses/metabolismo , TranscriptomaRESUMO
Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are widespread environmental contaminants linked to neuropsychological dysfunction in children. NDL PCBs increase spontaneous Ca(2+) oscillations in neurons by stabilizing ryanodine receptor (RyR) calcium release channels in the open configuration, which results in CREB-dependent dendritic outgrowth. In this study, we address the question of whether activation of CREB by NDL PCBs also triggers dendritic spine formation. Nanomolar concentrations of PCB 95, a NDL congener with potent RyR activity, significantly increased spine density and the frequency of miniature EPSCs in primary dissociated rat hippocampal cultures coincident with upregulation of miR132. Inhibition of RyR, CREB, or miR132 as well as expression of a mutant p250GAP cDNA construct that is not suppressed by miR132 blocked PCB 95 effects on spines and miniature EPSCs. PCB 95 also induced spine formation via RyR- and miR132-dependent mechanisms in hippocampal slice cultures. These data demonstrate a novel mechanism of PCB developmental neurotoxicity whereby RyR sensitization modulates spine formation and synaptogenesis via CREB-mediated miR132 upregulation, which in turn suppresses the translation of p250GAP, a negative regulator of synaptogenesis. In light of recent evidence implicating miR132 dysregulation in Rett syndrome and schizophrenia, these findings identify NDL PCBs as potential environmental risk factors for neurodevelopmental disorders.
Assuntos
Poluentes Ambientais/toxicidade , MicroRNAs/biossíntese , Neurogênese/fisiologia , Bifenilos Policlorados/toxicidade , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Neurogênese/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiologia , Sinapses/efeitos dos fármacosRESUMO
Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and ß-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, ß-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptin's induction of dendritic morphological changes that initiate spine and excitatory synapse formation.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Leptina/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Hipocampo/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
A subset of angiotensin IV (AngIV)-related molecules are known to possess procognitive/antidementia properties and have been considered as templates for potential therapeutics. However, this potential has not been realized because of two factors: 1) a lack of blood-brain barrier-penetrant analogs, and 2) the absence of a validated mechanism of action. The pharmacokinetic barrier has recently been overcome with the synthesis of the orally active, blood-brain barrier-permeable analog N-hexanoic-tyrosine-isoleucine-(6) aminohexanoic amide (dihexa). Therefore, the goal of this study was to elucidate the mechanism that underlies dihexa's procognitive activity. Here, we demonstrate that dihexa binds with high affinity to hepatocyte growth factor (HGF) and both dihexa and its parent compound Norleucine 1-AngIV (Nle(1)-AngIV) induce c-Met phosphorylation in the presence of subthreshold concentrations of HGF and augment HGF-dependent cell scattering. Further, dihexa and Nle(1)-AngIV induce hippocampal spinogenesis and synaptogenesis similar to HGF itself. These actions were inhibited by an HGF antagonist and a short hairpin RNA directed at c-Met. Most importantly, the procognitive/antidementia capacity of orally delivered dihexa was blocked by an HGF antagonist delivered intracerebroventricularly as measured using the Morris water maze task of spatial learning.
Assuntos
Angiotensina II/análogos & derivados , Cognição/fisiologia , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/metabolismo , Sinapses/metabolismo , Angiotensina II/metabolismo , Animais , Linhagem Celular , Cães , Células HEK293 , Hipocampo/metabolismo , Humanos , Células Madin Darby de Rim Canino , Masculino , Oligopeptídeos/metabolismo , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Angiotensin IV (AngIV: VYIHPF)-related peptides have long been recognized as procognitive agents with potential as antidementia therapeutics. Their development as useful therapeutics, however, has been limited by physiochemical properties that make them susceptible to metabolic degradation and impermeable to gut and blood-brain barriers. A previous study demonstrated that the core structural information required to impart the procognitive activity of the AngIV analog, norleucine(1)-angiotensin IV, resides in its three N-terminal amino acids, Nle-Tyr-Ile. The goal of this project was to chemically modify this tripeptide in such a way to enhance its metabolic stability and barrier permeability to produce a drug candidate with potential clinical utility. Initial results demonstrated that several N- and C-terminal modifications lead to dramatically improved stability while maintaining the capability to reverse scopolamine-induced deficits in Morris water maze performance and augment hippocampal synaptogenesis. Subsequent chemical modifications, which were designed to increase hydrophobicity and decrease hydrogen bonding, yielded an orally active, blood-barrier permeant, metabolically stabilized analog, N-hexanoic-Tyr-Ile-(6) aminohexanoic amide (dihexa), that exhibits excellent antidementia activity in the scopolamine and aged rat models and marked synaptogenic activity. These data suggest that dihexa may have therapeutic potential as a treatment of disorders, such as Alzheimer's disease, where augmented synaptic connectivity may be beneficial.
Assuntos
Angiotensina II/análogos & derivados , Demência/prevenção & controle , Nootrópicos/farmacologia , Oligopeptídeos/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Cromatografia Líquida de Alta Pressão , Espinhas Dendríticas/efeitos dos fármacos , Meia-Vida , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Ligação de Hidrogênio , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Neurogênese/efeitos dos fármacos , Oligopeptídeos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Espectrofotometria Ultravioleta , Sinapses/efeitos dos fármacos , TransfecçãoRESUMO
Hepatocyte growth factor (HGF), a neurotrophic protein, acting through its tyrosine kinase receptor, Met, facilitates learning and synaptic plasticity. In concert with the role of the HGF/Met system in synaptic plasticity, we demonstrate that Met is localized to brain regions which undergo extensive synaptic remodeling. We demonstrate that Met activation results in an increase in dendritic spine density and functional synapses. Based on these observations, we hypothesized that Met should be associated with post-synaptic elements found on dendritic spines. Thus, the goal of this study was to determine the sub-cellular localization of Met on hippocampal neurons. Using an atomic force microscopy tip decorated with a specific Met antibody, the location of Met was mapped to different cellular compartments of hippocampal pyramidal neurons. Our results indicated that multimeric activated Met was found to be concentrated in the dendritic compartment while the inactivated monomeric form of Met was prominent on the soma. FROM THE CLINICAL EDITOR: The goal of this study was to determine the sub-cellular localization of Met on hippocampal neurons using nanotechnology-based techniques, using an atomic force microscopy tip decorated with a specific Met antibody. The authors demonstrate that multimeric activated Met was found to be concentrated in the dendritic compartment while the inactivated monomeric form of Met was prominent in the soma of hippocampal pyramidal neurons.
Assuntos
Hipocampo/citologia , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Adesão Celular/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Fator de Crescimento de Hepatócito/farmacologia , Microscopia Confocal , Microscopia de Fluorescência , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacosRESUMO
Rett syndrome (RTT) is a severe neurodevelopmental disorder that arise from de novo mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Circulating levels of the adipocyte hormone leptin are elevated in RTT patients and rodent models of the disease. Leptin targets a large number of brain structures and regulates a wide range of developmental and physiological functions which are altered in RTT. We hypothesized that elevated leptin levels might contribute to RTT pathogenesis. Accordingly, we show that pharmacological antagonism of leptin or genetic reduction of leptin production prevents the degradation of health status, weight loss and the progression of breathing and locomotor deficits. At the neuronal level, the anti-leptin strategies rescue the hippocampal excitatory/inhibitory imbalance and synaptic plasticity impairment. Targeting leptin might therefore represent a new approach for RTT treatment.
RESUMO
The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during experience-dependent plasticity. This mini-symposium review will feature ongoing research into how spines are regulated by actin-signaling pathways during development and plasticity. It will also highlight evolving studies into how disruptions to these pathways might be functionally coupled to congenital disorders such as mental retardation.
Assuntos
Citoesqueleto/metabolismo , Espinhas Dendríticas/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Actinas/metabolismo , Animais , Microtúbulos/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Angiotensin IV (AngIV; Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6))-related peptides have emerged as potential antidementia agents. However, their development as practical therapeutic agents has been impeded by a combination of metabolic instability, poor blood-brain barrier permeability, and an incomplete understanding of their mechanism of action. This study establishes the core structure contained within norleucine(1)-angiotensin IV (Nle(1)-AngIV) that is required for its procognitive activity. Results indicated that Nle(1)-AngIV-derived peptides as small as tetra- and tripeptides are capable of reversing scopolamine-induced deficits in Morris water maze performance. This identification of the active core structure contained within Nle(1)-AngIV represents an initial step in the development of AngIV-based procognitive drugs. The second objective of the study was to clarify the general mechanism of action of these peptides by assessing their ability to affect changes in dendritic spines. A correlation was observed between a peptide's procognitive activity and its capacity to increase spine numbers and enlarge spine head size. These data suggest that the procognitive activity of these molecules is attributable to their ability to augment synaptic connectivity.
Assuntos
Hipocampo/crescimento & desenvolvimento , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Receptores de Angiotensina/química , Sinapses/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Cognição/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Antagonistas Muscarínicos/farmacologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Escopolamina/antagonistas & inibidores , Escopolamina/farmacologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Here we have used tandem mass spectrometry to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, we propose that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac.
Assuntos
Proteínas dos Microfilamentos/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dados de Sequência Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Família de Proteínas da Síndrome de Wiskott-AldrichRESUMO
Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synaptic plasticity remain largely uncharacterized. We show here that the CREB- and activity-regulated microRNA, miR132, is induced during periods of active synaptogenesis. Moreover, miR132 is necessary and sufficient for hippocampal spine formation. Expression of the miR132 target, p250GAP, is inversely correlated with miR132 levels and spinogenesis. Furthermore, knockdown of p250GAP increases spine formation while introduction of a p250GAP mutant unresponsive to miR132 attenuates this activity. Inhibition of miR132 decreases both mEPSC frequency and the number of GluR1-positive spines, while knockdown of p250GAP has the opposite effect. Additionally, we show that the miR132/p250GAP circuit regulates Rac1 activity and spine formation by modulating synapse-specific Kalirin7-Rac1 signaling. These data suggest that neuronal activity regulates spine formation, in part, by increasing miR132 transcription, which in turn activates a Rac1-Pak actin remodeling pathway.
Assuntos
Espinhas Dendríticas/fisiologia , MicroRNAs/metabolismo , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Bicuculina/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Espinhas Dendríticas/ultraestrutura , Antagonistas GABAérgicos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/citologia , MicroRNAs/genética , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2'O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.
Assuntos
Dendritos/metabolismo , Regulação para Baixo/genética , Proteínas Ativadoras de GTPase/genética , MicroRNAs/metabolismo , Plasticidade Neuronal , Transmissão Sináptica , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , MicroRNAs/genética , Biossíntese de Proteínas , Ratos , Ratos Sprague-DawleyRESUMO
Developing hippocampal neurons undergo rapid synaptogenesis in response to neurotrophic signals to form and refine circuit connections. The adipokine leptin is a satiety factor with neurotrophic actions, which potentiates both glutamatergic and GABAergic synaptogenesis in the hippocampus during neonatal development. Brief exposure to leptin enhances GABAA receptor-dependent synaptic currents in hippocampal neurons. Here, using molecular and electrophysiological techniques, we found that leptin increased the surface localization of GABAA receptors and the number of functional GABAergic synapses in hippocampal cultures from male and female rat pups. Leptin increased the interaction between GABAA receptors and the Rho guanine exchange factor ß-PIX (a scaffolding protein at GABAergic postsynaptic sites) in a manner dependent on the kinase CaMKK. We also found that the leptin receptor and ß-PIX formed a complex, the amount of which transiently increased upon leptin receptor activation. Furthermore, Tyr985 in the leptin receptor and the SH3 domain of ß-PIX are crucial for this interaction, which was required for the developmental increase in GABAergic synaptogenesis. Our results suggest a mechanism by which leptin promotes GABAergic synaptogenesis in hippocampal neurons and reveal further complexity in leptin receptor signaling and its interactome.
Assuntos
Leptina , Neurônios , Fatores de Troca de Nucleotídeo Guanina Rho , Animais , Feminino , Hipocampo/citologia , Leptina/metabolismo , Masculino , Neurônios/metabolismo , Ratos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Sinapses/metabolismoRESUMO
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. Mouse models of RTT show reduced expression of the cation-chloride cotransporter KCC2 and altered chloride homeostasis at presymptomatic stages. However, whether these alterations persist to late symptomatic stages has not been studied. Here we assess KCC2 and NKCC1 expressions and chloride homeostasis in the hippocampus of early [postnatal (P) day 30-35] and late (P50-60) symptomatic male Mecp2-null (Mecp2 -/y) mice. We found (i) no difference in the relative amount, but an over-phosphorylation, of KCC2 and NKCC1 between wild-type (WT) and Mecp2 -/y hippocampi and (ii) no difference in the inhibitory strength, nor reversal potential, of GABA A -receptor-mediated responses in Mecp2 -/y CA3 pyramidal neurons compared to WT at any stages studied. Altogether, these data indicate the presence of a functional chloride extrusion mechanism in Mecp2 -/y CA3 pyramidal neurons at symptomatic stages.
RESUMO
Members of the Wnt signaling family are important mediators of numerous developmental events, including activity-dependent dendrite development, but the pathways regulating expression and secretion of Wnt in response to neuronal activity are poorly defined. Here, we identify an NMDA receptor-mediated, Ca2+-dependent signaling pathway that couples neuronal activity to dendritic arborization through enhanced Wnt synthesis and secretion. Activity-dependent dendritic outgrowth and branching in cultured hippocampal neurons and slices is mediated through activation by CaM-dependent protein kinase kinase (CaMKK) of the membrane-associated gamma isoform of CaMKI. Downstream effectors of CaMKI include the MAP-kinase pathway of Ras/MEK/ERK and the transcription factor CREB. A serial analysis of chromatin occupancy screen identified Wnt-2 as an activity-dependent CREB-responsive gene. Neuronal activity enhances CREB-dependent transcription of Wnt-2, and expression of Wnt-2 stimulates dendritic arborization. This novel signaling pathway contributes to dynamic remodeling of the dendritic architecture in response to neuronal activity during development.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Dendritos/fisiologia , Transcrição Gênica/genética , Proteína Wnt2/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dendritos/enzimologia , Dendritos/genética , Ativação Enzimática/fisiologia , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Proteína Wnt2/genéticaRESUMO
Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca(2+)-signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca(2+)/calmodulin kinase kinase (CaMKK) and its downstream target Ca(2+)/calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the gamma-isoform of CaMKI as shRNA to CaMKIgamma, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKIgamma, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKIgamma as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKIgamma and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKIgamma, which subsequently promote axon formation.
Assuntos
Axônios/fisiologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Membrana Celular/metabolismo , Células Cultivadas , Microdomínios da Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neuritos/fisiologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Canais de Cátion TRPC/genéticaRESUMO
The canonical physiological role of leptin is to regulate hunger and satiety acting on specific hypothalamic nuclei. Beyond this key metabolic function; leptin also regulates many aspects of development and functioning of neuronal hippocampal networks throughout life. Here we show that leptin controls chloride homeostasis in the developing rat hippocampus in vitro. The effect of leptin relies on the down-regulation of the potassium/chloride extruder KCC2 activity and is present during a restricted period of postnatal development. This study confirms and extends the role of leptin in the ontogenesis of functional GABAergic inhibition and helps understanding how abnormal levels of leptin may contribute to neurological disorders.
Assuntos
Cloretos/metabolismo , Regulação para Baixo , Hipocampo/metabolismo , Homeostase , Leptina/farmacologia , Simportadores/metabolismo , Animais , Animais Recém-Nascidos , Regulação para Baixo/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Ratos Wistar , Cotransportadores de K e Cl-RESUMO
The ability to access intracellular targets is of vital importance as the number of identified druggable intracellular targets increases every year. However, intracellular delivery poses a formidable barrier, as many potential therapeutics are impermeable to cell membranes, which hinders their practical application in drug development. Herein we present de novo-designed unnatural cell penetrating peptide foldamers utilizing a 2,3-Didehydro-2-deoxyneuraminic acid (Neu2en) scaffold. Conveniently, this scaffold is amenable to standard Fmoc-based solid-phase peptide synthesis, with the advantages of tunable secondary structures and enhanced biostability. Flow cytometry and live-cell confocal microscopy studies showed that these Neu2en-based peptides, hereinafter termed SialoPen peptides, have significantly superior uptake in HeLa and primary neuronal hippocampal cells, outperforming the classical cell permeable peptides penetratin and HIV-TAT.
RESUMO
Activation of the leptin receptor, LepRb, by the adipocytokine/neurotrophic factor leptin in the central nervous system has procognitive and antidepressive effects. Leptin has been shown to increase glutamatergic synaptogenesis in multiple brain regions. In contrast, mice that have a mutation in the LepRb gene show abnormal synapse development in the hippocampus as well as deficits in cognition and increased depressive-like symptoms. Leptin increases glutamatergic synaptogenesis, in part, through enhancement of N-methyl-D-aspartic acid (NMDA) receptor function; yet the underlying signaling pathway is not known. In this study, we examine how leptin regulates surface expression of NR2B-containing NMDA receptors in hippocampal neurons. Leptin stimulation increases NR2BY1472 phosphorylation, which is inhibited by the Src family kinase inhibitor, PP1. Moreover, we show that Fyn, a member of the Src family kinases, is required for leptin-stimulated NR2BY1472 phosphorylation. Furthermore, inhibiting Y1472 phosphorylation with either a dominant negative Fyn mutant or an NR2B mutant that lacks the phosphorylation site (NR2BY1472F) blocks leptin-stimulated synaptogenesis. Additionally, we show that LepRb forms a complex with NR2B and Fyn. Taken together, these findings expand our knowledge of the LepRb interactome and the mechanisms by which leptin stimulates glutamatergic synaptogenesis in the developing hippocampus. Comprehending these mechanisms is key for understanding dendritic spine development and synaptogenesis, alterations of which are associated with many neurological disorders.