Dystroglycan Mediates Clustering of Essential GABAergic Components in Cerebellar Purkinje Cells.

Muscle dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane ß-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells.

Differential regulation of neurexin at glutamatergic and GABAergic synapses.

Neurexins (Nrxs) have emerged as potential determinants of synaptic specificity, but little is known about their localization at central synapses. Here we show that Nrxs have a remarkably selective localization at distinct types of glutamatergic synapses and we reveal an unexpected ontogenetic regulation of Nrx expression at GABAergic synapses. Our data indicate that synapses are specified by molecular interactions that involve both Nrx-dependent and Nrx-independent mechanisms. We propose that differences in the spatio-temporal profile of Nrx expression may contribute to specify the molecular identity of synapses.

Synaptic competition sculpts the development of GABAergic axo-dendritic but not perisomatic synapses.

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABAA receptors (GABAARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice ("γ2 knockdown mice") with an engineered allele of the GABAAR γ2 subunit gene which produced a mosaic expression of synaptic GABAARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABAARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABAARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABAARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.

Selective localization of collybistin at a subset of inhibitory synapses in brain circuits.

Collybistin is a brain-specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ-aminobutyric acid A receptors (GABA(A) Rs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous collybistin isoforms. We show that collybistin co-clusters with gephyrin and GABA(A) Rs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, collybistin is present in only a subset of gephyrin-positive synapses, with variable co-localization values in different brain regions. Moreover, collybistin co-localizes with GABA(A) Rs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABA(A) R subtypes. Surprisingly, we found that collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the collybistin gene in human X-linked intellectual disability and mouse knockout models.

Understanding the molecular diversity of GABAergic synapses.

GABAergic synapses exhibit a high degree of subcellular and molecular specialization, which contrasts with their apparent simplicity in ultrastructural appearance. Indeed, when observed in the electron microscope, GABAergic synapses fit in the symmetric, or Gray's type II category, being characterized by a relatively simple postsynaptic specialization. The inhibitory postsynaptic density cannot be readily isolated, and progress in understanding its molecular composition has lagged behind that of excitatory synapses. However, recent studies have brought significant progress in the identification of new synaptic proteins, revealing an unexpected complexity in the molecular machinery that regulates GABAergic synaptogenesis. In this article, we provide an overview of the molecular diversity of GABAergic synapses, and we consider how synapse specificity may be encoded by selective trans-synaptic interactions between pre- and postsynaptic adhesion molecules and secreted factors that reside in the synaptic cleft. We also discuss the importance of developing cataloguing tools that could be used to decipher the molecular diversity of synapses and to predict alterations of inhibitory transmission in the course of neurological diseases.

A new phenylalanine derivative acts as an antagonist at the AMPA receptor GluA2 and introduces partial domain closure: synthesis, resolution, pharmacology, and crystal structure.

In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and GluA2(R)(o) receptors with an approximately 4-fold preference for GluA1/2 vs GluA3/4. In TEVC electrophysiological experiments (RS)-3h competitively antagonized GluA2(Q)(i) receptors. The X-ray structure of the active enantiomer (S)-3h in complex with GluA2-S1S2J showed a domain closure around 8°. Even though the nitro and the carboxyethyl groups of (S)-3h were both anchored to Tyr702 through a water H-bond network, these interactions only induced weak subtype selectivity. In spite of the fact that (S)-3h induced a domain closure close to that observed for partial agonists, it did not produce agonist responses at GluA2 receptors under nondesensitizing conditions. 2-Carboxyethylphenylalanine derivatives provide a new synthetic scaffold for the introduction of substituents that could lead to AMPA receptor subtype-selective ligands.