Tue G. Banke

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and is associated with attention deficits, hyperactivity, anxiety, impulsivity, and repetitive behaviors. The disorder results from transcriptional silencing of the FMR1 gene, leading to loss of fragile X messenger ribonucleoprotein (FMRP), an RNA‐binding protein that regulates local dendritic translation by repressing ribosomal activity. To examine how impaired local protein synthesis affects dendritic organization, we used Golgi–Cox staining to analyze hippocampal CA1 principal neurons across four developmental stages (P14–21, P30–40, P60–80, and P120–150) in an FXS mouse model. We identified a progressive reduction in dendritic complexity, reflected by decreased Sholl intersections and reduced dendritic branch number and length. In contrast, spine density was increased in both apical and basal dendrites during early development but normalized to wild‐type levels in adulthood. Collectively, these structural alterations are likely to disrupt neural circuit development, with downstream consequences for cognition and behavior characteristic of FXS.

NMDA receptors (NMDARs) play important roles in synaptic transmission and are involved in multiple neurological disorders. During development, GluN2A in the forebrain becomes incorporated into previously GluN2B-dominated NMDARs, leading to the “GluN2A/GluN2B ratio switch.” This is a crucial step for normal brain development. Here we present findings indicating that GluN2A expression occurs earlier in the fragile X mouse and this could be an underlying factor contributing to the pathology found in the fragile X model.

AMPA-type glutamate receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits and play important roles in synaptic transmission and plasticity. Here, we have investigated the development of AMPAR-mediated synaptic transmission in the hippocampus of the Fmr1 knock-out (KO) mouse, a widely used model of Fragile X syndrome (FXS). FXS is the leading monogenic cause of intellectual disability and autism spectrum disorders (ASD) and it is considered a neurodevelopmental disorder. For that reason, we investigated synaptic properties and dendritic development in animals from an early stage when synapses are starting to form up to adulthood. We found that hippocampal CA1 pyramidal neurons in the Fmr1-KO mouse exhibit a higher AMPAR-NMDAR ratio early in development but reverses to normal values after P13. This increase was accompanied by a larger presence of the GluA2-subunit in synaptic AMPARs that will lead to altered Ca²⁺ permeability of AMPARs that could have a profound impact upon neural circuits, learning, and diseases. Following this, we found that young KO animals lack Long-term potentiation (LTP), a well-understood model of synaptic plasticity necessary for proper development of circuits, and exhibit an increased frequency of spontaneous miniature excitatory postsynaptic currents, a measure of synaptic density. Furthermore, post hoc morphological analysis of recorded neurons revealed altered dendritic branching in the KO group. Interestingly, all these anomalies are transitory and revert to normal values in older animals. Our data suggest that loss of FMRP during early development leads to temporary upregulation of the GluA2 subunit and this impacts synaptic plasticity and altering morphological dendritic branching.

Key points: Fragile X syndrome (FXS) is a genetic condition that is the most common form of inherited intellectual impairment and causes a range of neurodevelopmental complications including learning disabilities and intellectual disability and shares many characteristics with autism spectrum disorder (ASD). In the FXS mouse model, Fmr1^−/y, impaired synaptic plasticity was restored by pharmacologically inhibiting GluN2A‐containing NMDA receptors but not GluN2B‐containing receptors. Similar results were obtained by crossing Fmr1^−/y with GluN2A knock‐out (Grin2A^−/−) mice. These results suggest that dampening the elevated levels of GluN2A‐containing NMDA receptors in Fmr1^−/y mice has the potential to restore hyperexcitability of the neural circuitry to (a more) normal‐like level of brain activity. NMDA receptors (NMDARs) play important roles in synaptic plasticity at central excitatory synapses, and dysregulation of their function may lead to severe disorders such Fragile X syndrome (FXS). FXS is caused by transcriptional silencing of the FMR1 gene followed by lack of the encoding protein. Here we examined the effects of pharmacological and genetic manipulation of hippocampal NMDAR functions in long‐term potentiation (LTP) and depression (LTD). We found impaired NMDAR‐dependent LTP in the Fmr1‐deficient mice, which could be fully restored when GluN2A‐containing NMDARs was pharmacological inhibited. Interestingly, similar LTP effects were observed when the GluN2A gene (Grin2a) was deleted in Fmr1^−/y mice (Fmr1^−/y/Grin2a^−/− double knockout). In addition, GluN2A inhibition improved elevated mGluR5‐dependent LTD to normal level in the Fmr1^−/y mouse. These findings suggest that GluN2A is a promising target in FXS research that could help us better understand the disorder.

Fragile X syndrome (FXS) is a neurodevelopmental disease. It is one of the leading monogenic causes of intellectual disability among boys with most also displaying autism spectrum disorder traits. Here we investigated the role of NMDA receptors on mGluR-dependent long-term depression (mGluR-LTD), a key biomarker in the disease, at four different developmental stages. First, we applied the mGluR agonist 3,5-dihydroxyphenylglycine in the absence or presence of the NMDAR blocker, APV, hereby unmasking the NMDAR component in this process. As expected, in the presence of APV, we found more LTD in the mouse KO than in WT. This, however, was only observed in the p30–60 age group. At all other age groups tested, mGluR-LTD was almost identical between KO and WT. Interestingly, at p60, in the absence of APV, no or very little LTD was found in KO that was completely restored by application of APV. This suggests that the underlying cause of the enhanced mGluR-LTD in KO (at p30) is caused by dysregulated NMDAR signaling. To investigate this further, we next used NMDAR-subunit-specific antagonists. Inhibition of GluN2B, but not GluN2A, blocked mGluR-LTD only in WT. This was in contrast in the KO where blocking GluN2B rescued mGluR-LTD, suggesting GluN2B-containing NMDARs in the KO are hyperactive. Thus, these findings suggest strong involvement of GluN2B-containing-NMDARs in the pathophysiology of FXS and highlight a potential path for treatment for the disease.SIGNIFICANCE STATEMENTThere is currently no cure for fragile X, although medications targeting specific FXS symptoms do exist. The FXS animal model, theFmr1knock-out mouse, has demonstrated an increased mGluR5-mediated long-term depression (LTD) leading to several clinical trials of mGluR5 inhibitors/modulators, yet all have failed. In addition, surprisingly little information exists about the possible role of other ion channels/receptors, including NMDA receptors (NMDAR), in mGluR-LTD. Here we focus on NMDARs and their regulation of mGluR-mediated LTD at different developmental stages using several different NMDAR blockers/antagonists. Our findings suggest dysregulated NMDARs in the pathophysiology of FXS leading to altered mGluR-mediated LTD. Together, these data will help to develop new drug candidates that could lead to reversal of the FXS phenotype.

The transient receptor potential vanilloid 1 (TRPV1) channel is a non-selective cation channel that is mainly found in nociceptive neurons of the peripheral nervous system; however, these channels have also been located within the CNS, including the entorhinal cortex. Whole-cell patch-clamp recordings of principal entorhinal cortex (EC) layers II/III neurons revealed that evoked inhibitory postsynaptic currents were depressed by application of the TRPV1 agonist capsaicin (CAP), accompanied by a change in the pair-pulse ratio (PPR). In addition, recordings of miniature inhibitory postsynaptic currents (mIPSCs) revealed that inter-event intervals but not amplitude were decreased in wild-type (WT) after application of CAP. This suggests that TRPV1 channels are functional in the entorhinal cortex and are located on inhibitory neurons with their axonal arborization within layers II/III. In order to study TRPV1 channels and their involvement in long-term potentiation (LTP) induction in a more intact circuit, extracellular field potential recordings were performed in EC layers II/III. It was found that activated TRPV1 channels preclude induction of long-term potentiation. In sharp contrast, clear LTP was observed when antagonizing TRPV1 channels or recording from TRPV1 knock-out mice. Thus, these results suggests that signaling through activating inhibitory presynaptic TRPV1 channels represents a novel mechanism by which a shift in feed-forward inhibition of layers II/III cortical principal neurons prompt changes in synaptic strength and thereby contribute to a change of information storage within the brain.

The aims of this study were to investigate behaviour relevant to human autism spectrum disorder (ASD) and the fragile X syndrome in adolescent Fmr1 knockout (KO) mice and to evaluate the tissue levels of striatal monoamines. Fmr1 KO mice were evaluated in the open field, marble burying and three-chamber test for the presence of hyperactivity, anxiety, repetitive behaviour, sociability and observation of social novelty compared with wild-type (WT) mice. The Fmr1 KO mice expressed anxiety and hyperactivity in the open field compared with WT mice. This increased level of hyperactivity was confirmed in the three-chamber test. Fmr1 KO mice spent more time with stranger mice compared with the WT. However, after a correction for hyperactivity, their apparent increase in sociability became identical to that of the WT. Furthermore, the Fmr1 KO mice could not differentiate between a familiar or a novel mouse. Monoamines were measured by HPLC: Fmr1 KO mice showed an increase in the striatal dopamine level. We conclude that the fragile X syndrome model seems to be useful for understanding certain aspects of ASD and may have translational interest for studies of social behaviour when hyperactivity coexists in ASD patients.

CaMKII phosphorylates the GluA1 AMPA subunit at Ser831 to increase single channel conductance. Here, the authors show that coexpression of TARPs is required for phospho-Ser831 to increase conductance of GluA1/GluA2 receptors. Ser831 phosphorylation increases the efficiency with which each subunit can activate, increasing the likelihood of simultaneous subunit activation during gating. The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca2+/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.