Autism-related deficits via dysregulated eIF4E-dependent translational control.

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Investigation of genes important in neurodevelopment disorders in adult human brain.

Several neurodevelopmental disorders (NDDs) are caused by mutations in genes expressed in fetal brain, but little is known about these same genes in adult human brain. Here, we test the hypothesis that genes associated with NDDs continue to have a role in adult human brain to explore the idea that NDD symptoms may be partially a result of their adult function rather than just their neurodevelopmental function. To demonstrate adult brain function, we performed expression analyses and ChIPseq in human neural stem cell(NSC) lines at different developmental stages and adult human brain, targeting two genes associated with NDDs, SATB2 and EHMT1, and the WNT signaling gene TCF7L2, which has not been associated with NDDs. Analysis of DNA interaction sites in neural stem cells reveals high (40-50 %) overlap between proliferating and differentiating cells for each gene in temporal space. Studies in adult brain demonstrate that consensus sites are similar to NSCs but occur at different genomic locations. We also performed expression analyses using BrainSpan data for NDD-associated genes SATB2, EHMT1, FMR1, MECP2, MBD5, CTNND2, RAI1, CHD8, GRIN2A, GRIN2B, TCF4, SCN2A, and DYRK1A and find high expression of these genes in adult brain, at least comparable to developing human brain, confirming that genes associated with NDDs likely have a role in adult tissue. Adult function of genes associated with NDDs might be important in clinical disease presentation and may be suitable targets for therapeutic intervention.

Selective regulation of GluA subunit synthesis and AMPA receptor-mediated synaptic function and plasticity by the translation repressor 4E-BP2 in hippocampal pyramidal cells.

The eukaryotic initiation factor 4E-binding protein-2 (4E-BP2) is a repressor of cap-dependent mRNA translation and a major downstream effector of the mammalian target of rapamycin (mTOR) implicated in hippocampal long-term synaptic plasticity and memory. Yet, synaptic mechanisms regulated by 4E-BP2 translational repression remain unknown. Combining knock-out mice, whole-cell recordings, spine analysis, and translation profiling, we found that 4E-BP2 deletion selectively upregulated synthesis of glutamate receptor subunits GluA1 and GluA2, facilitating AMPA receptor (AMPAR)-mediated synaptic transmission and affecting translation-dependent chemically induced late long-term potentiation (cL-LTP). In 4E-BP2 knock-out (4E-BP2(-/-)) mice, evoked and miniature EPSCs were increased, an effect mimicked by short-hairpin RNA knockdown of 4E-BP2 in wild-type mice, indicating that 4E-BP2 level regulates basal transmission at mature hippocampal AMPAR-containing synapses. Remarkably, in 4E-BP2(-/-) mice, the AMPA to NMDA receptor (NMDAR) EPSC ratio was increased, without affecting NMDAR-mediated EPSCs. The enhanced AMPAR function concurred with increased spine density and decreased length resulting from greater proportion of regular spines and less filopodia in 4E-BP2(-/-) mice. Polysome profiling revealed that translation of GluA1 and GluA2 subunits, but not GluN1 or GluN2A/B, was selectively increased in 4E-BP2(-/-) hippocampi, consistent with unaltered I-V relation of EPSCs mediated by GluA1/GluA2 heteromers. Finally, translation-dependent cL-LTP of unitary EPSCs was also affected in 4E-BP2(-/-) mice, lowering induction threshold and removing mTOR signaling requirement while impairing induction by normal stimulation. Thus, translational control through 4E-BP2 represents a unique mechanism for selective regulation of AMPAR synthesis, synaptic function, and long-term plasticity, important for hippocampal-dependent memory processes.

Progranulin deficiency decreases gross neural connectivity but enhances transmission at individual synapses.

Frontotemporal dementia (FTD) has been linked to mutations in the progranulin gene (GRN) that lead to progranulin (PGRN) haploinsufficiency. Thus far, our understanding of the effects of PGRN depletion in the brain has been derived from investigation of gross pathology, and more detailed analyses of cellular function have been lacking. We report that knocking down PGRN levels in rat primary hippocampal cultures reduces neural connectivity by decreasing neuronal arborization and length as well as synapse density. Despite this, the number of synaptic vesicles per synapse and the frequency of mEPSCs are increased in PGRN knockdown cells, suggesting an increase in the probability of release at remaining synapses. Interestingly, we demonstrate that the number of vesicles per synapse is also increased in postmortem brain sections from FTD patients with PGRN haploinsufficiency, relative to controls. Our observations show that PGRN knockdown severely alters neuronal connectivity in vitro and that the synaptic vesicle phenotype observed in culture is consistent with that observed in the hippocampus of FTD patients.

IgSF21 promotes differentiation of inhibitory synapses via binding to neurexin2α.

Coordinated development of excitatory and inhibitory synapses is essential for higher brain function, and impairment in this development is associated with neuropsychiatric disorders. In contrast to the large body of accumulated evidence regarding excitatory synapse development, little is known about synaptic adhesion and organization mechanisms underlying inhibitory synapse development. Through unbiased expression screens and proteomics, we identified immunoglobulin superfamily member 21 (IgSF21) as a neurexin2α-interacting membrane protein that selectively induces inhibitory presynaptic differentiation. IgSF21 localizes postsynaptically and recruits axonal neurexin2α in a trans-interaction manner. Deleting IgSF21 in mice impairs inhibitory presynaptic organization, especially in the hippocampal CA1 stratum radiatum, and also diminishes GABA-mediated synaptic transmission in hippocampal CA1 neurons without affecting their excitatory synapses. Finally, mice lacking IgSF21 show a sensorimotor gating deficit. These findings suggest that IgSF21 selectively regulates inhibitory presynaptic differentiation through interacting with presynaptic neurexin2α and plays a crucial role in synaptic inhibition in the brain.Molecular mechanisms regulating the development of inhibitory synapses are poorly understood. Here the authors show that IgSF21 interacts with neurexin2α to induce presynaptic differentiation of inhibitory synapses, and that mice lacking IgSF21 exhibit deficits in inhibitory synaptic transmission.

A Rapid Pipeline to Model Rare Neurodevelopmental Disorders with Simultaneous CRISPR/Cas9 Gene Editing.

The development of targeted therapeutics for rare neurodevelopmental disorders (NDDs) faces significant challenges due to the scarcity of subjects and the difficulty of obtaining human neural cells. Here, we illustrate a rapid, simple protocol by which patient derived cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using an episomal vector and differentiated into neurons. Using this platform enables patient somatic cells to be converted to physiologically active neurons in less than two months with minimal labor. This platform includes a method to combine somatic cell reprogramming with CRISPR/Cas9 gene editing at single cell resolution, which enables the concurrent development of clonal knockout or knock-in models that can be used as isogenic control lines. This platform reduces the logistical barrier for using iPSC technology, allows for the development of appropriate control lines for use in rare neurodevelopmental disease research, and establishes a fundamental component to targeted therapeutics and precision medicine. Stem Cells Translational Medicine 2017;6:886-896.

Metaplastic Regulation of CA1 Schaffer Collateral Pathway Plasticity by Hebbian MGluR1a-Mediated Plasticity at Excitatory Synapses onto Somatostatin-Expressing Interneurons

Cortical GABAergic interneurons represent a highly diverse neuronal type that regulates neural network activity. In particular, interneurons in the hippocampal CA1 oriens/alveus (O/A-INs) area provide feedback dendritic inhibition to local pyramidal cells and express somatostatin (SOM). Under relevant afferent stimulation patterns, they undergo long-term potentiation (LTP) of their excitatory synaptic inputs through multiple induction and expression mechanisms. However, the cell-type specificity of these different forms of LTP and their specific contribution to the dynamic regulation of the CA1 network remain unclear. Here we recorded from SOM-expressing interneurons (SOM-INs) in the O/A region from SOM-Cre-Ai3 transgenic mice in whole-cell patch-clamp. Results indicate that, like in anatomically identified O/A-INs, theta-burst stimulation (TBS) induced a Hebbian form of LTP dependent on metabotropic glutamate receptor type 1a (mGluR1a) in SOM-INs, but not in parvalbumin-expressing interneurons, another mainly nonoverlapping interneuron subtype in CA1. In addition, we demonstrated using field recordings from transgenic mice expressing archaerhodopsin 3 selectively in SOM-INs, that a prior conditioning TBS in O/A, to induce mGluR1a-dependent LTP in SOM-INs, upregulated LTP in the Schaffer collateral pathway of pyramidal cells. This effect was prevented by light-induced hyperpolarization of SOM-INs during TBS, or by application of the mGluR1a antagonist LY367385, indicating a necessity for mGluR1a and SOM-INs activation. These results uncover that SOM-INs perform an activity-dependent metaplastic control on hippocampal CA1 microcircuits in a cell-specific fashion. Our findings provide new insights on the contribution of interneuron synaptic plasticity in the regulation of the hippocampal network activity and mnemonic processes.

Action-potential-independent GABAergic tone mediated by nicotinic stimulation of immature striatal miniature synaptic transmission.

Stimulation of presynaptic nicotinic acetylcholine receptors (nAChRs) increases the frequency of miniature excitatory synaptic activity (mEPSCs) to a point where they can promote cell firing in hippocampal CA3 neurons. We have evaluated whether nicotine regulation of miniature synaptic activity can be extended to inhibitory transmission onto striatal medium spiny projection neurons (MSNs) in acute brain slices. Bath application of micromolar nicotine typically induced 12-fold increases in the frequency of miniature inhibitory synaptic currents (mIPSCs). Little effect was observed on the amplitude of mIPSCs or mEPSCs under these conditions. Nicotine stimulation of mIPSCs was dependent on entry of extracellular calcium because removal of calcium from perfusate was able to block its action. To assess the potential physiological significance of the nicotine-stimulated increase in mIPSC frequency, we also examined the nicotine effect on evoked IPSCs (eIPSCs). eIPSCs were markedly attenuated by nicotine. This effect could be attributed to two potential mechanisms: transmitter depletion due to extremely high mIPSC rates and/or a reduction in presynaptic excitability associated with nicotinic depolarization. Treatment with low concentrations of K(+) was able to in part mimic nicotine's stimulatory effect on mIPSCs and inhibitory effect on eIPSCs. Current-clamp recordings confirmed a direct depolarizing action of nicotine that could dampen eIPSC activity leading to a switch to striatal inhibitory synaptic transmission mediated by tonic mIPSCs.

Effects of exercise on NMDA receptor subunit contributions to bidirectional synaptic plasticity in the mouse dentate gyrus.

We examined synaptic plasticity in the dentate gyrus (DG) of the hippocampus in vitro in juvenile C57Bl6 mice (28-40 days of age), housed in control conditions with minimal enrichment (Controls) or with access to an exercise wheel (Runners). LTP expression was significantly greater in slices from Runners than in those from Controls, but could be blocked by APV in both groups. LTP was significantly reduced by NR2B subunit antagonists in both groups. NVP-AAM077, an antagonist with a higher preference for NR2A subunits over NR2B subunits, blocked LTP in slices from Runners and produced a slight depression in Control animals. LTD in the DG was also blocked by APV, but not by either of the NR2B specific antagonists. Strikingly, NVP-AAM077 prevented LTD in Runners, but not in Control animals, suggesting an increased involvement of NR2A subunits in LTD in animals that exercise. NVP-AAM077 did not block LTD in NR2A Knock Out (KO) animals that exercised, as expected. In an attempt to discern whether NMDA receptors located at extrasynaptic sites could play a role in the induction of LTD, DL-TBOA was used to block excitatory amino acid transport and increase extracellular glutamate levels. Under these conditions, LTD was not blocked by the co-application of a specific NR2B subunit antagonist in either group, but NVP-AAM077 again blocked LTD selectively in Runners. These results indicate that NR2A and NR2B subunits play a significant role in LTP in the DG, and that exercise can significantly alter the contribution of NMDA NR2A subunits to LTD.