Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons

Abstract

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Dynamics and nanoscale organization of the postsynaptic endocytic zone at excitatory synapses

“…Thus, Itsn1 could have a central organizing role at the EZ. Consistently, we recently reported that Itsn1 knockdown abrogates mGluR-mediated AMPAR trafficking (van Gelder et al, 2020). The results presented here show that Itsn1 knockdown does not alter the location or overall morphology of the EZ indicating that while Itsn1 has likely an important role in coordinating endocytosis at the EZ, Itsn1 does not seem to directly support the maintenance of this clathrin structure.…”

“…GFP-Syndapin I was a gift from Dr. Robinson. GFP-Eps15 was a gift from Dr. Van Bergen en Henegouwen The following constructs have been described before: Homer1c-mCherry, Homer1c-GFP, Dynamin2-GFP (Scheefhals et al, 2019), pSM155-mirItsn-GFP (van Gelder et al, 2020) , GFP-CLCa knock-in construct (Willems et al, 2020).…”

Cerebral Expression of Metabotropic Glutamate Receptor Subtype 5 in Idiopathic Autism Spectrum Disorder and Fragile X Syndrome: A Pilot Study

“…FXS results from the presence of the fragile X mental retardation 1 ( FMR1 ) gene leading to deficits of Fragile X Mental Retardation Protein (FMRP). Dysregulated activation of group I metabotropic glutamate receptors [metabotropic glutamate receptors subtypes 1 and 5 (mGluR 1/5 )] causing metabotropic glutamate receptor dependent long-term depression (mGluR-LTD) plays a role in the pathogenesis of FXS [ 15 , 16 ]. The mechanisms of mGluR 1/5 dysregulation leading to the neurobehavioral symptoms of FXS have been elucidated by the study of fmr1 knockout (KO) mouse models.…”

A Picture Worth a Thousand Molecules—Integrative Technologies for Mapping Subcellular Molecular Organization and Plasticity in Developing Circuits

“…Using click chemistry labeling (Parker and Pratt, 2020), noncanonical AAs can be biotinylated for pull-down purification/mass spectrometry or fluorescently labeled for microscopic visualization. These approaches, called bio-orthogonal noncanonical amino acid tagging (BONCAT; Dieterich et al, 2006) and fluorescent noncanonical amino acid tagging (FUNCAT; Dieterich et al, 2010) respectively, have been used to measure changes in protein synthesis in response to neurotrophic factors (Dieterich et al, 2010;Genheden et al, 2015;Bowling et al, 2016), dopaminergic transmission (Hodas et al, 2012), activity-dependent homeostatic plasticity (Schanzenbächer et al, 2016(Schanzenbächer et al, , 2018, as well as the induction of long-term synaptic depression (Younts et al, 2016;van Gelder et al, 2020). BONCAT/FUNCAT have also been used to label whole organisms and demonstrate neural activity-dependent changes in protein translation during larval zebrafish (Hinz et al, 2012) and frog development (Shen et al, 2014;Liu and Cline, 2016;Liu et al, 2018).…”