Development Of Glutamatergic Synapses Research Articles

Regulation of AMPA receptor trafficking by secreted protein factors

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain. Regulation of AMPAR levels at synapses controls synaptic strength and underlies information storage and processing. Many proteins interact with the intracellular domain of AMPARs to regulate their trafficking and synaptic clustering. However, a growing number of extracellular factors important for glutamatergic synapse development, maturation and function have emerged that can also regulate synaptic AMPAR levels. This mini-review highlights extracellular protein factors that regulate AMPAR trafficking to control synapse development and plasticity. Some of these factors regulate AMPAR clustering and mobility by interacting with the extracellular N-terminal domain of AMPARs whereas others regulate AMPAR trafficking indirectly via their respective signaling receptors. While several of these factors are secreted from neurons, others are released from non-neuronal cells such as glia and muscle. Although it is apparent that secreted factors can act locally on neurons near their sites of release to coordinate individual synapses, it is less clear if they can diffuse over longer ranges to coordinate related synapses within a circuit or region of the brain. Given that there are hundreds of factors that can be secreted from neuronal and non-neuronal cells, it will not be surprising if more extracellular factors that modulate AMPARs and glutamatergic synapses are discovered. Many open questions remain including where and when the factors are expressed, what regulates their secretion from different cell types, what controls their diffusion, stability, and range of action, and how their cognate receptors influence intracellular signaling to control AMPAR trafficking.

Depletion of microglia in developing cortical circuits reveals its critical role in glutamatergic synapse development, functional connectivity, and critical period plasticity

Microglia populate the early developing brain and mediate pruning of the central synapses. Yet, little is known on their functional significance in shaping the developing cortical circuits. We hypothesize that the developing cortical circuits require microglia for proper circuit maturation and connectivity, and as such, ablation of microglia during the cortical critical period may result in a long-lasting circuit abnormality. We administered PLX3397, a colony-stimulating factor 1 receptor inhibitor, to mice starting at postnatal day 14 and through P28, which depletes >75% of microglia in the visual cortex (VC). This treatment largely covers the critical period (P19-32) of VC maturation and plasticity. Patch clamp recording in VC layer 2/3 (L2/3) and L5 neurons revealed increased mEPSC frequency and reduced amplitude, and decreased AMPA/NMDA current ratio, indicative of altered synapse maturation. Increased spine density was observed in these neurons, potentially reflecting impaired synapse pruning. In addition, VC intracortical circuit functional connectivity, assessed by laser scanning photostimulation combined with glutamate uncaging, was dramatically altered. Using two photon longitudinal dendritic spine imaging, we confirmed that spine elimination/pruning was diminished during VC critical period when microglia were depleted. Reduced spine pruning thus may account for increased spine density and disrupted connectivity of VC circuits. Lastly, using single-unit recording combined with monocular deprivation, we found that ocular dominance plasticity in the VC was obliterated during the critical period as a result of microglia depletion. These data establish a critical role of microglia in developmental cortical synapse pruning, maturation, functional connectivity, and critical period plasticity.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS.

CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

Targeted sequencing of the LRRTM gene family in suicide attempters with bipolar disorder.

Glutamatergic signaling is the primary excitatory neurotransmission pathway in the brain, and its relationship to neuropsychiatric disorders is of considerable interest. Our previous attempted suicide genome-wide association study, and numerous studies investigating gene expression, genetic variation, and DNA methylation have implicated aberrant glutamatergic signaling in suicide risk. The glutamatergic pathway gene LRRTM4 was an associated gene identified in our attempted suicide genome-wide association study, with association support seen primarily in females. Recent evidence has also shown that glutamatergic signaling is partly regulated by sex-related hormones. The LRRTM gene family encodes neuronal leucine-rich transmembrane proteins that localize to and promote glutamatergic synapse development. In this study, we sequenced the coding and regulatory regions of all four LRRTM gene members plus a large intronic region of LRRTM4 in 476 bipolar disorder suicide attempters and 473 bipolar disorder nonattempters. We identified two male-specific variants, one female- and five male-specific haplotypes significantly associated with attempted suicide in LRRTM4. Furthermore, variants within significant haplotypes may be brain expression quantitative trait loci for LRRTM4 and some of these variants overlap with predicted hormone response elements. Overall, these results provide supporting evidence for a sex-specific association of genetic variation in LRRTM4 with attempted suicide.

Tiam1 is Critical for Glutamatergic Synapse Structure and Function in the Hippocampus.

Mounting evidence suggests numerous glutamatergic synapse subtypes exist in the brain, and that these subtypes are likely defined by unique molecular regulatory mechanisms. Recent work has identified substantial divergence of molecular composition between commonly studied Schaffer collateral synapses and perforant path-dentate gyrus (DG) synapses of the hippocampus. However, little is known about the molecular mechanisms that may confer unique properties to perforant path-DG synapses. Here we investigate whether the RhoGEF (Rho guanine-nucleotide exchange factor) protein Tiam1 plays a unique role in the regulation of glutamatergic synapses in dentate granule neurons using a combination of molecular, electrophysiological, and imaging approaches in rat entorhino-hippocampal slices of both sexes. We find that inhibition of Tiam1 function in dentate granule neurons reduces synaptic AMPA receptor function and causes dendritic spines to adopt an elongated filopodia-like morphology. We also find that Tiam1's support of perforant path-DG synapse function is dependent on its GEF domain and identify a potential role for the auto-inhibitory PH domain of Tiam1 in regulating Tiam1 function at these synapses. In marked contrast, reduced Tiam1 expression in CA1 pyramidal neurons produced no effect on glutamatergic synapse development. Together, these data identify a critical role for Tiam1 in the hippocampus and reveal a unique Tiam1-mediated molecular program of glutamatergic synapse regulation in dentate granule neurons.SIGNIFICANCE STATEMENT Several lines of evidence independently point to the molecular diversity of glutamatergic synapses in the brain. Rho guanine-nucleotide exchange factor (RhoGEF) proteins as powerful modulators of glutamatergic synapse function have also become increasingly appreciated in recent years. Here we investigate the synaptic regulatory role of the RhoGEF protein Tiam1, whose expression appears to be remarkably enriched in granule neurons of the dentate gyrus. We find that Tiam1 plays a critical role in the development of glutamatergic perforant path-dentate gyrus synapses, but not in commonly studied in Schaffer collateral-CA1 synapses. Together, these data reveal a unique RhoGEF-mediated molecular program of glutamatergic synapse regulation in dentate granule neurons.

Apical-Basal Polarity Signaling Components, Lgl1 and aPKCs, Control Glutamatergic Synapse Number and Function.

SummaryNormal synapse formation is fundamental to brain function. We show here that an apical-basal polarity (A-BP) protein, Lgl1, is present in the postsynaptic density and negatively regulates glutamatergic synapse numbers by antagonizing the atypical protein kinase Cs (aPKCs). A planar cell polarity protein, Vangl2, which inhibits synapse formation, was decreased in synaptosome fractions of cultured cortical neurons from Lgl1 knockout embryos. Conditional knockout of Lgl1 in pyramidal neurons led to reduction of AMPA/NMDA ratio and impaired plasticity. Lgl1 is frequently deleted in Smith-Magenis syndrome (SMS). Lgl1 conditional knockout led to increased locomotion, impaired novel object recognition and social interaction. Lgl1+/− animals also showed increased synapse numbers, defects in open field and social interaction, as well as stereotyped repetitive behavior. Social interaction in Lgl1+/− could be rescued by NMDA antagonists. Our findings reveal a role of apical-basal polarity proteins in glutamatergic synapse development and function and also suggest a potential treatment for SMS patients with Lgl1 deletion.

Genetic Deletion of GABAA Receptors Reveals Distinct Requirements of Neurotransmitter Receptors for GABAergic and Glutamatergic Synapse Development.

In the adult brain GABAA receptors (GABAARs) mediate the majority of synaptic inhibition that provides inhibitory balance to excitatory drive and controls neuronal output. In the immature brain GABAAR signaling is critical for neuronal development. However, the cell-autonomous role of GABAARs in synapse development remains largely unknown. We have employed the CRISPR-CAS9 technology to genetically eliminate GABAARs in individual hippocampal neurons and examined GABAergic and glutamatergic synapses. We found that development of GABAergic synapses, but not glutamatergic synapses, critically depends on GABAARs. By combining different genetic approaches, we have also removed GABAARs and two ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), in single neurons and discovered a striking dichotomy. Indeed, while development of glutamatergic synapses and spines does not require signaling mediated by these receptors, inhibitory synapse formation is crucially dependent on them. Our data reveal a critical cell-autonomous role of GABAARs in inhibitory synaptogenesis and demonstrate distinct molecular mechanisms for development of inhibitory and excitatory synapses.

Insufficient glutamine synthetase activity during synaptogenesis causes spatial memory impairment in adult mice

Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.

Class 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus

To understand how proper circuit formation and function is established in the mammalian brain, it is necessary to define the genes and signaling pathways that instruct excitatory and inhibitory synapse development. We previously demonstrated that the ligand-receptor pair, Sema4D and Plexin-B1, regulates inhibitory synapse development on an unprecedentedly fast time-scale while having no effect on excitatory synapse development. Here, we report previously undescribed synaptogenic roles for Sema4A and Plexin-B2 and provide new insight into Sema4D and Plexin-B1 regulation of synapse development in rodent hippocampus. First, we show that Sema4a, Sema4d, Plxnb1, and Plxnb2 have distinct and overlapping expression patterns in neurons and glia in the developing hippocampus. Second, we describe a requirement for Plexin-B1 in both the presynaptic axon of inhibitory interneurons as well as the postsynaptic dendrites of excitatory neurons for Sema4D-dependent inhibitory synapse development. Third, we define a new synaptogenic activity for Sema4A in mediating inhibitory and excitatory synapse development. Specifically, we demonstrate that Sema4A signals through the same pathway as Sema4D, via the postsynaptic Plexin-B1 receptor, to promote inhibitory synapse development. However, Sema4A also signals through the Plexin-B2 receptor to promote excitatory synapse development. Our results shed new light on the molecular cues that promote the development of either inhibitory or excitatory synapses in the mammalian hippocampus.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation.

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses

Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. In this work, we have expressed the human DISC1 gene in the fruit fly (Drosophila melanogaster) and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. We found that DISC1 interacts with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1–mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.

Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway

Development of functional synapses is crucial for the transmission and storage of information in the brain. Post establishment of the initial synaptic contact, synapses are stabilized through neuronal activity-induced signals. Emerging studies have implicated ubiquitination; a reversible posttranslational modification, as a key regulatory switch that modulates synapse development through proteasomal degradation. Ubiquitination of proteins is precisely regulated by E3 ligases, a set of enzymes that bind to specific substrates to facilitate the conjugation of monomeric or polymeric ubiquitin. However, the identity of specific E3 ubiquitin ligases that influence activity-dependent maturation of synapses and the mechanism by which ubiquitination of proteins regulate functional synapse development remain elusive. Here, we have identified a RING domain containing E3 ligase, Rnf2, asan activity-regulated factor that modulates glutamatergic synapse development in the hippocampus. Rnf2 is a synapse associated E3 ligase that is stabilized by neuronal activity through self-polyubiquitination. We have shown that neuronal activity shifts the balance toward stabilization of Rnf2 through self-polyubiquitination rather than triggering its degradation through polyubiquitination by Ube3A, an E3 ligase implicated in Angelman Syndrome. Our synapse density measurements and whole-cell patch-clamp recordings have revealed that the loss of Rnf2 function in cultured hippocampal neurons result in the development of ‘silent’ synapses that lack GluA1 containing functional AMPA receptors. These results provide a plausible mechanistic approach toward understanding how synapse maturation is regulated via the activity-dependent stabilization of Rnf2 through a non-canonical function of polyubiquitination.

Structural and functional insights into the latrophilin3 ‐ FLRT3‐UNC5D complex that mediates glutamatergic synapse development

Latrophilins (LPHNs) are neuronal adhesion‐type G‐protein coupled receptors able to establish cell‐surface interactions with other proteins through their characteristically large ectodomain. Fibronectin leucine‐rich repeat transmembrane (FLRT) proteins constitute such endogenous ligands and the LPHN3‐FLRT3 trans‐cellular interaction has been shown to play an important role in glutamatergic synapse development in vitro and in vivo. With the use of a series of biochemical and biophysical techniques including analytical ultra‐centrifugation, deuterium‐exchange mass spectrometry, isothermal titration calorimetry and protein crystallography, we have performed an extensive characterization of the LPHN3‐FLRT3 interaction. In particular, 1) we have defined the binding domain of each partner to be the olfactomedin (OLF) domain in LPHN3 and the Leucin‐Rich Repeats (LRR) in FLRT3, 2) we have determined the crystal structure of the LPHN3‐OLF in two crystal forms, and 3) we have solved the crystal structure of the complex between LPHN3‐OLF and FLRT3‐LRR. In addition, 4) we have recently described higher‐ordered complexes formed by the LPHN‐FLRT sub‐complex and UNC5D, a receptor known to be implicated in axon guidance. Finally, 5) we are currently trying to identify novel ligands for some of these proteins. This work will enable us to understand, at the molecular level, the complex interplay of these proteins and their role in axon guidance and synapse formation.

Deficiency of Cks1 Leads to Learning and Long-Term Memory Defects and p27 Dependent Formation of Neuronal Cofilin Aggregates.

In mitotic cells, the cyclin-dependent kinase (CDK) subunit protein CKS1 regulates S phase entry by mediating degradation of the CDK inhibitor p27. Although mature neurons lack mitotic CDKs, we found that CKS1 was actively expressed in post-mitotic neurons of the adult hippocampus. Interestingly, Cks1 knockout ( Cks1−/− ) mice exhibited poor long-term memory, and diminished maintenance of long-term potentiation in the hippocampal circuits. Furthermore, there was neuronal accumulation of cofilin-actin rods or cofilin aggregates, which are associated with defective dendritic spine maturation and synaptic loss. We further demonstrated that it was the increased p27 level that activated cofilin by suppressing the RhoA kinase-mediated inhibitory phosphorylation of cofilin, resulting in the formation of cofilin aggregates in the Cks1−/− neuronal cells. Consistent with reports that the peptidyl-prolyl-isomerase PIN1 competes with CKS1 for p27 binding, we found that inhibition of PIN1 diminished the formation of cofilin aggregates through decreasing p27 levels, thereby activating RhoA and increasing cofilin phosphorylation. Our results revealed that CKS1 is involved in normal glutamatergic synapse development and dendritic spine maturation in adult hippocampus through modulating p27 stability.

Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development

Latrophilins (LPHNs) are adhesion-like G-protein-coupled receptors implicated in attention-deficit/hyperactivitydisorder. Recently, LPHN3 was found to regulate excitatory synapse number through trans interactions with fibronectin leucine-rich repeat transmembrane 3 (FLRT3). By isothermal titration calorimetry, we determined that only the olfactomedin (OLF) domain of LPHN3 is necessary for FLRT3 association. By multi-crystal native single-wavelength anomalous diffraction phasing, we determined the crystal structure of the OLF domain. This structure is a five-bladed β propeller with a Ca(2+) ion bound in the central pore, which is capped by a mobile loop that allows the ion to exchange with the solvent. The crystal structure of the OLF/FLRT3 complex shows that LPHN3-OLF in the closed state binds with high affinity to the concave face of FLRT3-LRR with a combination of hydrophobic and charged residues. Our study provides structural and functional insights into the molecular mechanism underlying the contribution of LPHN3/FLRT3 to the development of glutamatergic synapses.

Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

Gating Characteristics Control Glutamate Receptor Distribution and Trafficking In Vivo

Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking invivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.

The Specific α-Neurexin Interactor Calsyntenin-3 Promotes Excitatory and Inhibitory Synapse Development

Perturbations of cell surface synapse-organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-β) as a synapse-organizing protein unique in binding and recruiting α-neurexins, but not β-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3-shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3⁻/⁻ mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.

FLRT Proteins Are Endogenous Latrophilin Ligands and Regulate Excitatory Synapse Development

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.

Arrested Glutamatergic Synapse Development in Human Partial Epilepsy

While studying the brain function of the human partial epilepsy gene, leucine-rich glioma-inactivated 1 (LGI1), a new mechanism of human epileptogenesis was revealed-persistent immaturity of glutamatergic circuitries. LGI1, a novel secreted protein, was found to be increased during the postnatal period; when glutamatergic synapses both downregulate their presynaptic vesicular release probability and reduce their postsynaptic NMDA-receptor subunit NR2B. During this same period, the dendritic arbor and spines are pruned and remodeled. Using bacterial artificial chromosome transgenic mouse techniques, excess wild-type LGI1 was shown to magnify these critical brain developmental events in the hippocampal dentate gyrus; while an epilepsy-associated, truncated, dominant-negative form of LGI1 blocked them. By contrast, the hippocampal dentate granule neuron GABAergic synapses and intrinsic excitability were unaltered. A role for LGI1 in downregulating glutamate synapse function was confirmed by germline gene deletion; this intervention also revealed a selective increase of glutamatergic synaptic transmission with unaltered GABAergic synapses and intrinsic excitability of hippocampal CA1 pyramidal neurons. Interestingly, the role of LGI1 in neurological disease was further expanded when a subset of patients with limbic encephalitis (an autoimmune disorder with memory loss in 100% and seizures in 80% of individuals) were discovered to carry autoantibodies to LGI1.