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1.
Development ; 147(24)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33168583

RESUMO

The endocannabinoid (eCB) system, via the cannabinoid CB1 receptor, regulates neurodevelopment by controlling neural progenitor proliferation and neurogenesis. CB1 receptor signalling in vivo drives corticofugal deep layer projection neuron development through the regulation of BCL11B and SATB2 transcription factors. Here, we investigated the role of eCB signalling in mouse pluripotent embryonic stem cell-derived neuronal differentiation. Characterization of the eCB system revealed increased expression of eCB-metabolizing enzymes, eCB ligands and CB1 receptors during neuronal differentiation. CB1 receptor knockdown inhibited neuronal differentiation of deep layer neurons and increased upper layer neuron generation, and this phenotype was rescued by CB1 re-expression. Pharmacological regulation with CB1 receptor agonists or elevation of eCB tone with a monoacylglycerol lipase inhibitor promoted neuronal differentiation of deep layer neurons at the expense of upper layer neurons. Patch-clamp analyses revealed that enhancing cannabinoid signalling facilitated neuronal differentiation and functionality. Noteworthy, incubation with CB1 receptor agonists during human iPSC-derived cerebral organoid formation also promoted the expansion of BCL11B+ neurons. These findings unveil a cell-autonomous role of eCB signalling that, via the CB1 receptor, promotes mouse and human deep layer cortical neuron development.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Neurônios/metabolismo , Receptor CB1 de Canabinoide/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Proliferação de Células/efeitos dos fármacos , Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Endocanabinoides/agonistas , Endocanabinoides/genética , Endocanabinoides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Transdução de Sinais/genética
2.
J Neurosci ; 41(38): 7924-7941, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34353897

RESUMO

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.


Assuntos
Encéfalo/metabolismo , Canabinoides/metabolismo , Neurônios GABAérgicos/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Animais , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Knockout , Receptor CB1 de Canabinoide/genética
3.
J Neurosci ; 40(45): 8604-8617, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33046543

RESUMO

The second messenger cAMP is an important determinant of synaptic plasticity that is associated with enhanced neurotransmitter release. Long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell (PC) synapses depends on a Ca2+-induced increase in presynaptic cAMP that is mediated by Ca2+-sensitive adenylyl cyclases. However, the upstream signaling and the downstream targets of cAMP involved in these events remain poorly understood. It is unclear whether cAMP generated by ß-adrenergic receptors (ßARs) is required for PF-PC LTP, although noradrenergic varicosities are apposed in PF-PC contacts. Guanine nucleotide exchange proteins directly activated by cAMP [Epac proteins (Epac 1-2)] are alternative cAMP targets to protein kinase A (PKA) and Epac2 is abundant in the cerebellum. However, whether Epac proteins participate in PF-PC LTP is not known. Immunoelectron microscopy demonstrated that ßARs are expressed in PF boutons. Moreover, activation of these receptors through their agonist isoproterenol potentiated synaptic transmission in cerebellar slices from mice of either sex, an effect that was insensitive to the PKA inhibitors (H-89, KT270) but that was blocked by the Epac inhibitor ESI 05. Interestingly, prior activation of these ßARs occluded PF-PC LTP, while the ß1AR antagonist metoprolol blocked PF-PC LTP, which was also absent in Epac2-/- mice. PF-PC LTP is associated with an increase in the size of the readily releasable pool (RRP) of synaptic vesicles, consistent with the isoproterenol-induced increase in vesicle docking in cerebellar slices. Thus, the ßAR-mediated modulation of the release machinery and the subsequent increase in the size of the RRP contributes to PF-PC LTP.SIGNIFICANCE STATEMENT G-protein-coupled receptors modulate the release machinery, causing long-lasting changes in synaptic transmission that influence synaptic plasticity. Nevertheless, the mechanisms underlying synaptic responses to ß-adrenergic receptor (ßAR) activation remain poorly understood. An increase in the number of synaptic vesicles primed for exocytosis accounts for the potentiation of neurotransmitter release driven by ßARs. This effect is not mediated by the canonical protein kinase A pathway but rather, through direct activation of the guanine nucleotide exchange protein Epac by cAMP. Interestingly, this ßAR signaling via Epac is involved in long term potentiation at cerebellar granule cell-to-Purkinje cell synapses. Thus, the pharmacological activation of ßARs modulates synaptic plasticity and opens therapeutic opportunities to control this phenomenon.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Potenciação de Longa Duração/fisiologia , Receptores Adrenérgicos beta/fisiologia , Vesículas Sinápticas/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Cerebelo/citologia , Cerebelo/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Células de Purkinje/fisiologia , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura
4.
Artif Organs ; 45(10): 1183-1188, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33560549

RESUMO

High glutamate levels after head trauma or cerebral ischemia have neurotoxic effects. The objective of the present study was to evaluate the efficacy of hemodialysis to remove glutamate from the blood and to assess the behavior of this small molecule. Ten patients with end-renal disease on hemodialysis were included in the study. Glutamate clearance was evaluated within the first hour of hemodialysis on a midweek dialysis day on five patients who underwent low flux hemodialysis, whereas the other five patients underwent highly efficient hemodialysis (high flux hemodialysis on one day and online hemodiafiltration on another day). Glutamate clearance with hemodialysis was very effective and did not show any differences between the techniques (low flux: 214 [55], high flux: 204 [37], online hemodiafiltration: 202 [16], median (interquartile range), P = .7). Glutamate clearance was almost equivalent to vascular access plasma flow and it was not affected by dialyzer permeability or ultrafiltration rate. After a hemodialysis session, a significant decrease in glutamate blood level was observed (prehemodialysis: 59.7 [36.1], posthemodialysis 37.0 [49.2], P = .005). Dialysis performed under fasting condition showed higher glutamate reduction rate (60%) than that under feeding condition (20%). Hemodialysis may be an effective method to reduce glutamate blood levels, and the molecule clearance does not differ between the different techniques used. Considering previous results in experimental models, hemodialysis without hemodynamic stress, could be considered for reducing glutamate neurotoxic effects in acute ischemic strokes of patients in chronic hemodialysis programs.


Assuntos
Ácido Glutâmico/metabolismo , Hemodiafiltração/métodos , Diálise Renal/métodos , Idoso , Isquemia Encefálica/terapia , Jejum/sangue , Feminino , Ácido Glutâmico/sangue , Humanos , AVC Isquêmico/terapia , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade
5.
Neurobiol Dis ; 130: 104482, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31129085

RESUMO

In fragile X syndrome, the absence of Fragile X Mental Retardation Protein (FMRP) is known to alter postsynaptic function, although alterations in presynaptic function also occur. We found that the potentiation of glutamate release induced by the ß adrenergic receptor (ßAR) agonist isoproterenol is absent in cerebrocortical nerve terminals (synaptosomes) from mice lacking FMRP (Fmr1 KO), despite the normal cAMP generation. The glutamate release induced by moderate stimulation of synaptosomes with 5 mM KCl was not potentiated in Fmr1 KO synaptosomes by isoproterenol, nor by stimulating the receptor associated signaling pathway with the adenylyl cyclase activator forskolin or with the Epac activator 8-pCPT. Hence, the impairment in the pathway potentiating release is distal to ßARs. Electron microscopy shows that Fmr1 KO cortical synapses have more docked vesicles than WT synapses, consequently occluding the isoproterenol response through which more SVs approach the active zone (AZ) of the plasma membrane. Weak stimulation of synaptosomes with the Ca2+ ionophore ionomycin recovered the release potentiation driven by forskolin and 8-pCPT but not with isoproterenol, revealing an impairment in the efficiency of receptor generated cAMP to activate the release potentiation pathway. Indeed, inhibiting cyclic nucleotide phosphodiesterase PDE2A with BAY 60-7550 reestablished isoproterenol mediated potentiation in Fmr1 KO synaptosomes. Thus, the lack of ß-AR mediated potentiation of glutamate release appears to be the consequence of an impaired capability of the receptor to mobilize SVs to the AZ and because of a decreased efficiency of cAMP to activate the signaling pathway that enhances neurotransmitter release.


Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Ácido Glutâmico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinaptossomos/metabolismo
6.
Cereb Cortex ; 28(1): 307-322, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29121220

RESUMO

The vast majority of neurons within the striatum are GABAergic medium spiny neurons (MSNs), which receive glutamatergic input from the cortex and thalamus, and form two major efferent pathways: the direct pathway, expressing dopamine D1 receptor (D1R-MSNs), and the indirect pathway, expressing dopamine D2 receptor (D2R-MSNs). While molecular mechanisms of MSN degeneration have been identified in animal models of striatal damage, the molecular factors that dictate a selective vulnerability of D1R-MSNs or D2R-MSNs remain unknown. Here, we combined genetic, chemogenetic, and pharmacological strategies with behavioral and neurochemical analyses, and show that the pool of cannabinoid CB1 receptor (CB1R) located on corticostriatal terminals efficiently safeguards D1R-MSNs, but not D2R-MSNs, from different insults. This cell-specific response relies on the regulation of glutamatergic signaling, and is independent from the CB1R-dependent control of astroglial activity in the striatum. These findings define cortical CB1R as a pivotal synaptic player in dictating a differential vulnerability of D1R-MSNs versus D2R-MSNs, and increase our understanding of the role of coordinated cannabinergic-glutamatergic signaling in establishing corticostriatal circuits and its dysregulation in neurodegenerative diseases.


Assuntos
Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Vetores Genéticos , Ácido Glutâmico/metabolismo , Humanos , Proteína Huntingtina/administração & dosagem , Proteína Huntingtina/genética , Proteína Huntingtina/toxicidade , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Receptor CB1 de Canabinoide/genética , Transmissão Sináptica/fisiologia
7.
J Physiol ; 596(5): 921-940, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29280494

RESUMO

KEY POINTS: Neurotransmitter release is inhibited by metabotropic glutamate type 7 (mGlu7 ) receptors that reduce Ca2+ influx, yet synapses lacking this receptor also produce weaker release, suggesting that mGlu7 receptors may also prime synaptic vesicles for release. Prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs through a presynaptic effect. The inhibitory response is blocked by pertussis toxin, while the potentiating response is prevented by a phospholipase C inhibitor (U73122) and an inhibitor of diacylglycerol (DAG) binding (calphostin C), suggesting that this receptor also couples to pathways that generate DAG. Release potentiation is associated with an increase in the number of synaptic vesicles close to the plasma membrane, which was dependent on the Munc13-2 and RIM1α proteins. The Glu7 receptors activated by the glutamate released following high frequency stimulation provoke a bidirectional modulation of synaptic transmission. ABSTRACT: Neurotransmitter release is driven by Ca2+ influx at synaptic boutons that acts on synaptic vesicles ready to undergo exocytosis. Neurotransmitter release is inhibited when metabotropic glutamate type 7 (mGlu7 ) receptors provoke a reduction in Ca2+ influx, although the reduced release from synapses lacking this receptor suggests that they may also prime synaptic vesicles for release. These mGlu7 receptors activate phospholipase C (PLC) and generate inositol trisphosphate, which in turn releases Ca2+ from intracellular stores and produces diacylglycerol (DAG), an activator of proteins containing DAG-binding domains such as Munc13 and protein kinase C (PKC). However, the full effects of mGlu7 receptor signalling on synaptic transmission are unclear. We found that prolonged activation of mGlu7 receptors with the agonist l-AP4 first reduces and then enhances the amplitude of EPSCs, a presynaptic effect that changes the frequency but not the amplitude of the mEPSCs and the paired pulse ratio. Pertussis toxin blocks the inhibitory response, while the PLC inhibitor U73122, and the inhibitor of DAG binding calphostin C, prevent receptor mediated potentiation. Moreover, this DAG-dependent potentiation of the release machinery brings more synaptic vesicles closer to the active zone plasma membrane in a Munc13-2- and RIM1α-dependent manner. Electrically evoked release of glutamate that activates mGlu7 receptors also bidirectionally modulates synaptic transmission. In these conditions, potentiation now occurs rapidly and it overcomes any inhibition, such that potentiation prevails unless it is suppressed with the PLC inhibitor U73122.


Assuntos
Região CA1 Hipocampal/fisiologia , Diglicerídeos/metabolismo , Ácido Glutâmico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Animais , Proteínas de Ligação ao GTP/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naftalenos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Toxina Pertussis/farmacologia , Transdução de Sinais , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores
8.
J Neurochem ; 141(5): 662-675, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28295320

RESUMO

The recycling of synaptic vesicle (SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex (AP-1/AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. Cover Image for this issue: doi. 10.1111/jnc.13801.


Assuntos
Brefeldina A/farmacologia , Cerebelo/citologia , Endocitose/efeitos dos fármacos , Neurônios/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Vesículas Sinápticas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Endocitose/fisiologia , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Compostos de Piridínio/metabolismo , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacocinética , Ratos , Ratos Wistar , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Fatores de Tempo , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo
9.
J Neurochem ; 142(3): 350-364, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28445587

RESUMO

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.


Assuntos
Cerebelo/citologia , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Cerebelo/efeitos dos fármacos , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estrenos/farmacologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Pirrolidinonas/farmacologia , Ratos Wistar , Receptor CB1 de Canabinoide/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Fosfolipases Tipo C/metabolismo
10.
Proc Natl Acad Sci U S A ; 111(22): 8257-62, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24843137

RESUMO

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.


Assuntos
Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Neurônios/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Idoso , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Córtex Cerebral/citologia , Corpo Estriado/citologia , Endocanabinoides/metabolismo , Endocanabinoides/fisiologia , Endocanabinoides/uso terapêutico , Feminino , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Ácido Glutâmico/metabolismo , Humanos , Integrases/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Neurotoxinas/metabolismo , Técnicas de Cultura de Órgãos , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptores de GABA-A/metabolismo , Sinaptossomos/fisiologia
11.
Int J Mol Sci ; 18(11)2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084181

RESUMO

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (cGK) signaling pathway regulates the clustering and the recruitment of proteins and vesicles to the synapse, thereby adjusting the exoendocytic cycle to the intensity of activity. Accordingly, this pathway can accelerate endocytosis following large-scale exocytosis, and pre-synaptic cGK type II (cGKII) plays a major role in this process, controlling the homeostatic balance of vesicle exocytosis and endocytosis. We have studied synaptic vesicle recycling in cerebellar granule cells from mice lacking cGKII under strong and sustained stimulation, combining imaging techniques and ultrastructural analyses. The ultrastructure of synapses in the adult mouse cerebellar cortex was also examined in these animals. The lack of cGKII provokes structural changes to synapses in cultured cells and in the cerebellar cortex. Moreover, endocytosis is slowed down in a subset of boutons in these cells when they are stimulated strongly. In addition, from the results obtained with the selective inhibitor of cGKs, KT5823, it can be concluded that cGKI also regulates some aspects of vesicle cycling. Overall, these results confirm the importance of the cGMP pathway in the regulation of vesicle cycling following strong stimulation of cerebellar granule cells.


Assuntos
Cerebelo/citologia , Cerebelo/metabolismo , GMP Cíclico/metabolismo , Neurônios/metabolismo , Proteínas Quinases/metabolismo , Membranas Sinápticas/metabolismo , Animais , Endocitose , Exocitose , Imunofluorescência , Potenciais da Membrana , Camundongos , Camundongos Knockout , Imagem Molecular , Neurônios/ultraestrutura , Proteínas Quinases/genética , Vesículas Secretórias/metabolismo , Membranas Sinápticas/ultraestrutura
12.
J Neurosci ; 34(26): 8788-99, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-24966379

RESUMO

From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.


Assuntos
Cerebelo/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/genética
13.
J Biol Chem ; 288(43): 31370-85, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24036110

RESUMO

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the ß-adrenergic receptor (ßAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of ßARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that ßARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.


Assuntos
Córtex Cerebral/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos beta/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Adjuvantes Imunológicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Córtex Cerebral/citologia , Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , Isoproterenol/farmacologia , Camundongos , Naftalenos/farmacologia , Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Membranas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
14.
Biochim Biophys Acta ; 1833(8): 1820-31, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23545413

RESUMO

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.


Assuntos
Cerebelo/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/enzimologia , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Feminino , Neurônios/enzimologia , Fosforilação/genética , Ratos , Ratos Wistar , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
15.
J Cell Sci ; 125(Pt 2): 422-34, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22331355

RESUMO

Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles. Decreasing the extent of exocytosis dramatically increased the proportion of synaptic boutons that exhibited strong FM1-43-unloading and dramatically reduced the number of endosome-like structures. Hence, we concluded that efficient recycling of synaptic vesicles is concomitant with the formation of non-releasable endosomes in both types of synaptic boutons, although to different extents. Furthermore, cell maturation in culture increased the proportion of synaptic boutons that were capable of an intense release response, whereas the chronic blockage of synaptic activity diminished the capacity of boutons to release dye.


Assuntos
Endossomos/metabolismo , Exocitose , Vesículas Sinápticas/metabolismo , Animais , Inibidores de Calcineurina , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Dinaminas/fisiologia , Endocitose , Feminino , Corantes Fluorescentes , Masculino , Terminações Pré-Sinápticas/classificação , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio , Compostos de Amônio Quaternário , Ratos , Ratos Wistar , Vesículas Sinápticas/efeitos dos fármacos , Tacrolimo/farmacologia
16.
BMC Neurosci ; 14: 127, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24138605

RESUMO

BACKGROUND: In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis. METHODS: We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention. RESULTS: In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone. CONCLUSIONS: Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.


Assuntos
Imuno-Histoquímica/métodos , Neurônios/fisiologia , Imagem Óptica/métodos , Transmissão Sináptica/fisiologia , Animais , Cerebelo/fisiologia , Corantes Fluorescentes , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio , Compostos de Amônio Quaternário
17.
Mol Autism ; 14(1): 14, 2023 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-37029391

RESUMO

BACKGROUND: Fragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment. METHODS: We performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4. RESULTS: We found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by ß adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, ß adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice. LIMITATIONS: We cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells. CONCLUSIONS: Our study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.


Assuntos
Síndrome do Cromossomo X Frágil , Potenciação de Longa Duração , Masculino , Camundongos , Animais , Potenciação de Longa Duração/fisiologia , Síndrome do Cromossomo X Frágil/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Transmissão Sináptica , Modelos Animais de Doenças , Comportamento Social , Camundongos Knockout
18.
Sci Adv ; 9(25): eadf6222, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37343100

RESUMO

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N-ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.


Assuntos
Transmissão Sináptica , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Fusão de Membrana , Membrana Celular/metabolismo , Neurotransmissores/metabolismo
19.
Nat Commun ; 14(1): 2303, 2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37085487

RESUMO

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.


Assuntos
Canabinoides , Camundongos , Animais , Canabinoides/farmacologia , Canabinoides/metabolismo , Proteômica , Hipocampo/metabolismo , Transmissão Sináptica , Sinapses/metabolismo , Receptores de Canabinoides/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo
20.
Biochim Biophys Acta ; 1813(1): 14-26, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21056598

RESUMO

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK(+)) medium, and we have evaluated the effect of decreasing the [K(+)] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K(+)] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K(+)] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.


Assuntos
Cerebelo/metabolismo , Potenciais da Membrana , Receptores de AMPA/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citologia , Feminino , Técnicas Imunoenzimáticas , Masculino , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Subunidades Proteicas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Receptores de AMPA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo
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