Abstract
The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.
Highlights
These multiprotein complexes are organised into a hierarchy, and the most abundant postsynaptic supercomplex at vertebrate excitatory synapses is formed by PSD-95.5-8 Through its multiple protein–protein binding domains, PSD-95 is a central organiser at the postsynaptic density (PSD) of excitatory synapses, directly anchoring the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) at the membrane and assembling a network of proteins around the NMDAR to enable synaptic signalling.[9,10]
We found that interactions between genotype × test significantly affected the performance of Syngap[1] (F(1,12) = 15.59; p = 0.0019), Nlgn[3] (F(1,30) = 9.63; p = 0.0042) and Dlgap[1] (F(2,28) = 3.55; p = 0.0423) cohorts, with post hoc tests indicating that Syngap[1] and Nlgn[3] mutant mice achieved similar levels of accuracy to WT littermate controls in visual discrimination, but their performance was significantly different in reversal (Syngap1+/− mice displayed lower accuracy compared with WTs, and Nlgn3−/Y mice showed higher accuracy compared with WTs)
Our data show that gene mutations in interacting proteins of the NMDAR-PSD-95 complex lead to specific changes in different measures of learning and reaction times that underlie cognitive processing (Figure 6)
Summary
The postsynaptic terminal of excitatory synapses in vertebrate species contains a highly conserved set of proteins, including neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes that are tightly organised into multiprotein complexes - the signalling machinery essential for synaptic transmission and plasticity underlying the regulation of behaviour.[1,2,3,4,5] These multiprotein complexes are organised into a hierarchy, and the most abundant postsynaptic supercomplex at vertebrate excitatory synapses is formed by PSD-95.5-8 Through its multiple protein–protein binding domains, PSD-95 is a central organiser at the postsynaptic density (PSD) of excitatory synapses, directly anchoring the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) at the membrane and assembling a network of proteins around the NMDAR to enable synaptic signalling.[9,10] These interactors include cell adhesion molecules, such as neuroligins, numerous scaffold proteins, including DLGAP/GKAP and Shank, and various downstream cytoplasmic proteins, such as SynGAP, a GTPaseactivating protein (GAP) for Ras.[11,12,13,14,15] A large-scale mouse genetic screen of loss-of-function mutations in postsynaptic proteins showed that mutations in PSD-95 and its close interacting proteins had the strongest phenotypes in synaptic electrophysiology and behaviour, indicating that PSD-95 protein complexes are critical components of the postsynaptic terminal of excitatory synapses.[16,17] While many studies have investigated changes in measures of synaptic signalling and plasticity following mutations in genes encoding postsynaptic proteins, we know less about their roles in complex cognitive behaviour, especially given physiological phenotypes do not always map directly to distinct behavioural measures (e.g., impaired long-term potentiation does not always predict learning performance).[18]. We have previously shown that mice lacking the Dlg[4] gene, which encodes PSD-95, show robust impairments in simple associative learning,[37] whereas PSD-95 heterozygous mice display enhanced performance in the pairwise visual discrimination and reversal learning touchscreen tests.[41] Previous work by us and others has examined mice carrying mutations in NMDAR subunits in these same behavioural tests and shown that substitution of the GRIN2B intracellular C-terminal domain with GRIN2A,38 complete loss of GRIN2A42 or loss of GRIN2B-containing NMDARs on GABAergic interneurons[43] impaired visual discrimination, but did not impact flexibility in reversal learning These data provide tantalising evidence that distinct molecular components of the NMDAR-PSD-95 protein complex are differentially required for regulating discrimination and reversal learning.
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