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1.
Mol Psychiatry ; 26(12): 7596-7609, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34331007

RESUMO

Shank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.


Assuntos
Transtorno Autístico , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Modelos Animais de Doenças , Endossomos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo
2.
J Clin Invest ; 130(3): 1168-1184, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32039920

RESUMO

Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.


Assuntos
Corpo Estriado/metabolismo , Sistema de Sinalização das MAP Quinases , Neurônios/metabolismo , Doença de Parkinson Secundária/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Corpo Estriado/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Complexos Multiproteicos/agonistas , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neurônios/patologia , Doença de Parkinson Secundária/tratamento farmacológico , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/patologia , Ratos , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética
3.
Neuropharmacology ; 115: 92-99, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27276689

RESUMO

The orphan Glutamate receptor Delta2 (GluD2) intrinsic ion channel activity is indirectly triggered by glutamate through stimulation of the metabotropic glutamate receptor 1 (mGlu1), in cerebellar Purkinje cells. However, the mechanisms of GluD2 ion channel opening are entirely unknown. In this work, we investigated the signaling pathways underlying the mGlu1-induced GluD2 current, performing whole-cell voltage-clamp recordings from mGlu1 and GluD2 transfected HEK293 cells. We show that the activation of GluD2 channels via DHPG-induced mGlu1 stimulation is Gαq-dependent. Moreover, inhibition of the downstream components of the mGlu1 canonical signaling pathway PLC and PKC with U73122 and GF109203X, respectively, strongly reduced the DHPG-induced GluD2 current. These results were further confirmed on endogenous receptors at the Parallel Fiber - Purkinje Cell cerebellar synapse, indicating that the opening of the GluD2 channel by mGlu1 receptor mobilizes the canonical Gq-PLC-PKC pathway. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.


Assuntos
Receptores de Glutamato/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Transdução de Sinais/fisiologia , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Estrenos/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Pirrolidinonas/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
4.
Sci Rep ; 6: 28231, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27302735

RESUMO

Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Análise de Célula Única/métodos , Células Cultivadas , Células HEK293 , Humanos , Ligação Proteica , Transdução de Sinais
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