Efeitos da interação entre o Regulador de Crescimento Neuronal 1 e o Receptor Metabotrópico de Glutamato 5 sobre a sinalização de cálcio e a sobrevivência neuronal

Abstract

Glutamate is the primary excitatory neurotransmitter in the central nervous system of mammals. It acts on ionotropic (iGluRs) and metabotropic (mGluRs) receptors. Type 5 metabotropic glutamate receptor (mGluR5), through the activation of the Gαq/11-βγ protein, promotes the release of Ca2+ from intracellular stores, leading to the activation of extracellular signal-regulated kinase (ERK1/2), which is essential for neuronal survival. NEGR1 is a cell adhesion molecule crucial for neuronal arborization and maturation and is capable of activating ERK1/2. Our research group has demonstrated the physical interaction between mGluR5 and NEGR1. This study aims to evaluate the effects of the interaction between mGluR5 and NEGR1 on Ca2+ signaling and neuronal survival dependent on glutamate receptors. For this purpose, primary cultures of C57/Bl6 mouse neurons were transduced with lentivirus containing either shControl or shNEGR1, which promotes NEGR1 knockdown. The variation in [Ca2+]i was assessed upon stimulation with a selective mGluR5 agonist, (DHPG, 10 µM) or glutamate (50 µM). DHPG promoted an increase in [Ca2+]i, but no difference was observed between shControl and shNEGR1 neurons. Upon glutamate stimulation, the Ca2+ response was significantly lower in shNEGR1 neurons compared to shControl. The N-Methyl-DAspartate receptor (NMDAR) is an iGluR permeable to Ca2+. For this reason, the negative allosteric modulator of mGluR5, CTEP, and the NMDAR antagonist, MK-801, were used. CTEP and MK-801, individually or in combination, abolished the difference in [Ca2+]i observed between shNEGR1 and shControl neurons. These results suggest that NEGR1 participates in Ca2+ signaling dependent on mGluR5 and NMDAR when both are simultaneously active but does not interfere with the selective activation of mGluR5. To evaluate neuronal survival, neurons were subjected to a glutamate insult (50 µM) for 24 hours, mimicking an excitotoxicity condition. An increase in cell death was observed in shNEGR1 neurons compared to shControl. Complementary experiments were performed in mGluR5-/- primary neuronal cultures. In shNEGR1 neurons, the increase in neuronal death was significantly higher compared to shControl, demonstrating that this difference is independent of mGluR5. The data presented suggest that NEGR1 can modulate glutamate receptors, Ca2+ signaling, and neuronal death. The observed effect involves mGluR5 and NMDAR. Therefore, NEGR1 emerges as a potential target for investigating Ca2+ homeostasis and related dysfunctions. Further studies are crucial to elucidate the intracellular consequences of NEGR1 interaction with glutamate receptors.