Experience-induced remodeling of the hippocampal post-synaptic proteome and phosphoproteome

The post synaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the PSD proteome and phosphoproteome in mice following various treatments. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated for quantitative proteomic and phosphoproteomics analysis. We used a sequential enrichment strategy to explore the concurrent events of protein expression and phosphorylation in the hippocampal PSD following IA training (IA) or immediate shock (Shock). We identified more than 6,200 proteins and 3,000 phosphoproteins in the sequential strategy covering a total of 7,429 proteins. On the phosphoproteins we identified a total of 9,589 phosphosites. Strikingly, of the significantly IA-regulated proteins and phosphoproteins, a large fraction of the proteins displayed an overall decrease in phosphorylation level. Bioinformatic analysis of proteins and phosphoproteins that were regulated by IA were annotated for an involvement in regulation of glutamate receptor functionality, calcium signaling, and synaptic plasticity. We also identified synaptic kinases, phosphatases and their respective phosphosites regulated by IA training or iimmediate shock. Furthermore, we found that AMPA receptor surface expression was regulatedby protein phosphatase, Mg2+/Mn2+ dependent 1H (Ppm1h). Together, these results unravel the dynamic remodeling of the PSD upon IA learning or immediate shock and serve as a resource for elucidating the synaptic proteome dynamics induced by experience-dependent plasticity.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD

Here we report that sustained activation of GluK2 subunit-containing kainate receptors leads to AMPA receptor endocytosis and a novel form of long-term depression (KAR-LTDAMPAR) in hippocampal neurons. The KAR-evoked loss of surface AMPA receptors requires KAR channel activity and is occluded by the blockade of PKC or PKA. Moreover, in acute hippocampal slices, kainate invoked LTD of AMPA EPSCs. These data, together with our previously reported KAR-LTPAMPAR, demonstrate that KARs bidirectionally regulate synaptic AMPARs and synaptic plasticity.

A novel bioassay for quantification of surface Cannabinoid receptor 1 expression

The cannabinoid receptor type 1 (CB1) plays critical roles in multiple physiological processes such as pain perception, brain development and body temperature regulation. Mutations on this gene (CNR1), results in altered functionality and/or biosynthesis such as reduced membrane expression, changes in mRNA stability or changes in downstream signaling that act as triggers for diseases such as obesity, Parkinson’s, Huntington’s, among others; thus, it is considered as a potential pharmacological target. To date, multiple quantification methods have been employed to determine how these mutations affect receptor expression and localization; however, they present serious disadvantages that may arise quantifying errors. Here, we describe a sensitive bioassay to quantify receptor surface expression; in this bioassay the Gaussia Luciferase (GLuc) was fused to the extracellular portion of the CB1. The GLuc activity was assessed by coelenterazine addition to the medium followed by immediate readout. Based on GLuc activity assay, we show that the GLuc signals corelate with CB1 localization, besides, we showed the assay’s functionality and reliability by comparing its results with those generated by previously reported mutations on the CNR1 gene and by using flow cytometry to determine the cell surface receptor expression. Detection of membrane-bound CB1, and potentially other GPCRs, is able to quickly screen for receptor levels and help to understand the effect of clinically relevant mutations or polymorphisms.

Abnormal AMPAR-mediated synaptic plasticity, cognitive and autistic-like behaviors in a missense Fmr1 mutant mouse model of Fragile X syndrome

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. FXS is usually caused by a CGG-repeat expansion in the FMR1 gene leading to its silencing and the loss-of-expression of the Fragile X Mental Retardation Protein (FMRP). Missense mutations were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS in these patients, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that the Fmr1R138Q hippocampus has an increased spine density associated with postsynaptic ultrastructural defects and increased AMPA receptor surface expression. Combining biochemical assays, high-resolution imaging and electrophysiological recordings, we also show that the mutation impairs the hippocampal long-term potentiation (LTP) and leads to socio-cognitive deficits in Fmr1R138Q mice. These findings reveal that the R138Q mutation impacts the synaptic functions of FMRP and highlight potential mechanisms causing FXS in FMRP-R138Q patients.