INTERACTION WITH NEGR1 AFFECTS MGLU5 CELL SIGNALING AND THE NEUROPROTECTION INDUCED BY THE POSITIVE ALLOSTERIC MODULATOR VU0409551

Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that play an important role in cellular excitability and plasticity in the nervous system. Previous studies have shown that mGlu5 can activate other cell signaling pathways through interactions with different protein partners, including Homer proteins. Moreover, mGlu5 may interact with extracellular proteins, such as cellular prion protein (PrPc) which leads to intracellular calcium levels and the phosphorylation of receptors and proteins. In this study, we investigated a novel mglu5-interacting protein with the neuronal growth factor (NEGR1) and evaluated its role in receptor signaling and neuroprotection. Using nanoLC-MS/MS we identified a total of 21 proteins, including NEGR1, to be enriched in hippocampal samples immunoprecipitated with an anti-mGluR5 antibody from mGluR5+/+ mice compared to mGluR5-/- mice (n=6, p<0.05). The result was later validated with co-immunoprecipitation experiments (n=3). Primary neuronal cultures were prepared from E15 mouse embryos (CEUA: 50/2021). The functional role of NEGR1 was investigated using Lentivirus-delivery shRNA-Negr1-GFP at DIV 04, followed by experiments conducted at DIV 09. For the control group, we use a scramble shRNA. NEGR1 knockdown did not alter total mGlu5 expression or its plasma membrane localization, but reduced mGlu5 association with the synaptic scaffold protein Homer and the proportion of the receptor localized near the PSD95 (n=5, p<0.05). NEGR1 knockdown also impaired mGlu5- dependent ERK1/2 activation and reduced mRNA levels of Arc/Arg3.1 and BDNF (n=5, p<0.05). In contrast, DHPG-induced intracellular Ca²⁺ elevation was not affected by NEGR1 knockdown, suggesting that canonical mGlu5-Gαq/11 signalling is preserved (n=7). To evaluate mGlu5-dependent neuroprotection, neuronal cultures were exposed to 50 µM glutamate for 12 hr. Treatment with 1 µM VU0409551 prevented glutamate-induced neuronal death and neurite degeneration in control neurons, whereas NEGR1 knockdown abolished these protective effects (n=5, p<0.05).These findings demonstrate that NEGR1 is an important extracellular partner regulating mGlu5 synaptic localization and downstream signalling and is required for the neuroprotective effects exerted by VU0409551.

This work was supported by FAPEMIG, and CNPq