NEURONAL GROWTH REGULATOR 1 REGULATES NEURONAL EXCITABILITY AND MGLUR5 SIGNALING

Introduction: Metabotropic glutamate receptors (mGluR) are G protein-coupled receptors that play an important role in cellular excitability and plasticity in several regions of the nervous system. Previous studies have shown that mGluR5 may interacts with extracellular proteins, such as cellular prion protein (PrPc). Recently, we demonstrated that mGluR5 interacts with extracellular proteins of cell adhesion molecules (CAMs), belonging to the immunoglobulin superfamily (IgSF), including neuronal growth factor (Negr1). Moreover, Negr1, can modulate the signaling pathway triggered by mGluR5. In this study, we aim to elucidate the impact of the interaction between Negr1 and mGluR5. Methods: Females GD15 mice were used to prepare primary neuronal cultures (CEUA: 50/2021). To assess whether NEGR1 absence disrupt mGluR5 signaling, we develop a tool based on Lentivirus-delivery shRNA-Negr1-GFP, capable to silence NEGR1 expression in neurons culture. For control group, we use a scramble shRNA. Calcium imaging and Western blot were performed on DIV 9. Whole-Cell current clamp recordings were performed on DIV 9-15. Neuronal excitability was assessed by measuring the resting potential, the rheobase current at -75 mV, and the firing frequency using 1-4x Rb stimulus. Results: NEGR1 KD reduced calcium transients evoked by glutamate (50 µM) but did not change the response of the mGluR5 agonist, DHPG (10 µM) (n=18, p<0.05). Despite this, NEGR1 KD inhibits mGluR5-dependent ERK1/2 activation (N=4; p<0.05), confirming that NEGR1 may modulate mGluR5 signaling. The resting potential of control cultured neurons was -66.4 ± 3.0 mV and was not affected by NEGR1 KD (n=10, p= 0.20). Similarly, the rheobase was 28.2 ± 6.6 pA in the control group, with no significant difference in NEGR1 KD (n=5, p=0.22). However, Negr1 KD increased action potential firing frequency (n=10; p<0.05). Conclusions: Our findings indicate a role for NEGR1 in coupling mGluR5 to the ERK1/2 pathway. In addition, Negr1 KD increases neuronal excitability. Considering that, NEGR1 is crucial to synaptogenesis and the importance of mGluR5 in the brain, studying this interaction become relevant. Further studies are needed to identify the mechanisms behind the observed effects.