Phosphoproteomic Alterations of Ionotropic Glutamate Receptors in the Hippocampus of the Ts65Dn Mouse Model of Down Syndrome

Abstract

Down syndrome (DS), the main genetic cause of intellectual disability, is associated with an imbalance of excitatory/inhibitory neurotransmitter systems. The phenotypic assessment and pharmacotherapy interventions in DS murine models strongly pointed out glutamatergic neurotransmission alterations (specially affecting ionotropic glutamate receptors [iGluRs]) that might contribute to DS pathophysiology, which is in agreement with DS condition. iGluRs play a critical role in fast-mediated excitatory transmission, a process underlying synaptic plasticity. Neuronal plasticity is biochemically modulated by post-translational modifications, allowing rapid and reversible adaptation of synaptic strength. Among these modifications, phosphorylation/dephosphorylation processes strongly dictate iGluR protein–protein interactions, cell surface trafficking, and subsynaptic mobility. Hence, we hypothesized that dysregulation of phosphorylation/dephosphorylation balance might affect neuronal function, which in turn could contribute to the glutamatergic neurotransmitter alterations observed in DS. To address this point, we biochemically purified subsynaptic hippocampal fractions from adult Ts65Dn mice, a trisomic mouse model recapitulating DS phenotypic alterations. Proteomic analysis showed significant alterations of the molecular composition of subsynaptic compartments of hippocampal trisomic neurons. Further, we characterized iGluR phosphopattern in the hippocampal glutamatergic synapse of trisomic mice. Phosphoenrichment-coupled mass spectrometry analysis revealed specific subsynaptic- and trisomy-associated iGluR phosphorylation signature, concomitant with differential subsynaptic kinase and phosphatase composition of Ts65Dn hippocampal subsynaptic compartments. Furthermore, biochemical data were used to build up a genotype-kinome-iGluR phosphopattern matrix in the different subsynaptic compartments. Overall, our results provide a precise profile of iGluR phosphopattern alterations in the glutamatergic synapse of the Ts65Dn mouse model and support their contribution to DS-associated synaptopathy. The alteration of iGluR phosphoresidues in Ts65Dn hippocampi, together with the kinase/phosphatase signature, identifies potential novel therapeutic targets for the treatment of glutamatergic dysfunctions in DS.

The C‐terminal domains of the NMDA receptor: How intrinsically disordered tails affect signalling, plasticity and disease

“…NMDAR stimulation simultaneously activates phosphatases and phosphoproteomic studies showed that LTP causes similar numbers of sites to be phosphorylated and dephosphorylated (Li et al., 2016). The regulated phospho‐sites are span receptors, scaffolding proteins and transcription factors, and thus reorganize both the synaptic structure and downstream signalling (Coba, 2019) and may be dysregulated in neurological disorders (Gómez de Salazar, Grau, Ciruela, & Altafaj, 2018).…”

Endogenous AT1 receptor–protein kinase C activity in the hypothalamus augments glutamatergic input and sympathetic outflow in hypertension

“…3381, Cell Signaling Technology, Danvers, MA, USA) (Gomez de Salazar et al . ), a rabbit anti‐phospho‐GluN1 Ser 896 antibody (1:500, no. ABN88, EMD Millipore; Billerica, MA, USA) (Xie et al .…”

Ts65Dn synaptic proteome partially recapitulates AD biochemical signature

“…Postmortem EC from human subjects was selected for our study because EC in the medial temporal lobe is already affected in the early stages of AD (Braak and Braak, 1991). The cortical subsynaptic fractionation protocol was previously validated by our group by western blotting using specific subsynaptic compartment markers (Gomez de Salazar et al, 2018).…”

“…However, this study found only modest changes in the levels of synaptic proteins in the Ts65Dn mouse model. Therefore, we used proteomic analyses to study the postsynaptic and extrasynaptic fractions of the hippocampus of Ts65Dn mice (Gómez de Salazar et al, 2018) to avoid the confounding effect of mixing different brain regions.…”