Marco Sassoè-Pognetto

The γ2 Subunit of GABAA Receptors Is a Substrate for Palmitoylation by GODZ

The neurotransmitter GABA activates heteropentameric GABAA receptors, which are composed mostly of α, β, and γ2 subunits. Regulated membrane trafficking and subcellular targeting of GABAA receptors is important for determining the efficacy of GABAergic inhibitory function. Of special interest is the γ2 subunit, which is mostly dispensable for assembly and membrane insertion of functional receptors but essential for accumulation of GABAA receptors at synapses. In a search for novel receptor trafficking proteins, we have used the SOS-recruitment system and isolated a Golgi-specific DHHC zinc finger protein (GODZ) as a novel γ2 subunit-interacting protein. GODZ is a member of the superfamily of DHHC cysteine-rich domain (DHHC-CRD) polytopic membrane proteins shown recently in yeast to represent palmitoyltransferases. GODZ mRNA is found in many tissues; however, in brain the protein is detected in neurons only and highly concentrated and asymmetrically distributed in the Golgi complex. GODZ interacts with a cysteine-rich 14-amino acid domain conserved specifically in the large cytoplasmic loop of γ1-3 subunits but not in other GABAA receptor subunits. Coexpression of GODZ and GABAA receptors in heterologous cells results in palmitoylation of the γ2 subunit in a cytoplasmic loop domain-dependent manner. Neuronal GABAA receptors are similarly palmitoylated. Thus, GODZ-mediated palmitoylation represents a novel posttranslational modification that is selective forγ subunit-containing GABAA receptor subtypes, a mechanism that is likely to be important for regulated trafficking of these receptors in the secretory pathway.

Synaptic inhibition of Purkinje cells mediates consolidation of vestibulo-cerebellar motor learning

Although feedforward inhibition onto Purkinje cells was first documented 40 years ago, we understand little of how inhibitory interneurons contribute to cerebellar function in behaving animals. Using a mouse line (PC-Δγ2) in which GABAA receptor–mediated synaptic inhibition is selectively removed from Purkinje cells, we examined how feedforward inhibition from molecular layer interneurons regulates adaptation of the vestibulo-ocular reflex. Although impairment of baseline motor performance was relatively mild, the ability to adapt the phase of the vestibulo-ocular reflex and to consolidate gain adaptations was strongly compromised. Purkinje cells showed abnormal patterns of simple spikes, both during and in the absence of evoked compensatory eye movements. On the basis of modeling our experimental data, we propose that feedforward inhibition, by controlling the fine-scale patterns of Purkinje cell activity, enables the induction of plasticity in neurons of the cerebellar and vestibular nuclei.

Colocalization of multiple GABAA receptor subtypes with gephyrin at postsynaptic sites

Clustering of gamma aminobutyric acid (GABA)A receptors to postsynaptic sites requires the presence of both the γ2 subunit and gephyrin. Here, we analyzed by double‐immunofluorescence staining the colocalization of gephyrin and major GABAA‐receptor subtypes distinguished by the subunits α1, α2, α3, or γ2 in adult rat brain. By using confocal laser scanning microscopy, GABAA‐receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin‐positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABAA‐receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin‐positive clusters were not colocalized with GABAA‐receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High‐resolution images (voxel size, 50 × 50 × 150 nm) were restored with an iterative image deconvolution procedure based on a measured point‐spread function to analyze the colocalization between GABAA‐receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro‐organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin‐ and γ2 subunit‐positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were “embedded” in larger GABAA‐receptor clusters. These findings show that gephyrin is associated with a majority of GABAA‐receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy. J. Comp. Neurol. 420:481–498, 2000.

Learning, AMPA receptor mobility and synaptic plasticity depend on n‐cofilin‐mediated actin dynamics

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin‐binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n‐cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long‐term potentiation and long‐term depression. Loss of n‐cofilin‐mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n‐cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina

Neuroligins (NL1–NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimulus-coding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.

Mini-review: gephyrin, a major postsynaptic protein of GABAergic synapses.

γ‐aminobutyric acid type A (GABAA) receptors are located at the majority of inhibitory synapses in the mammalian brain. However, the mechanisms by which GABAA receptor subunits are targeted to, and clustered in, the postsynaptic membrane are poorly understood. Recent studies have demonstrated that gephyrin, a protein first identified as a component of the glycine receptor (GlyR) complex, is colocalized with several subtypes of GABAA receptors and is involved in the stabilization of postsynaptic GABAA receptor clusters. Thus, gephyrin functions as a clustering protein for major subtypes of inhibitory ion channel receptors.

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty‐seeking behavior

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE‐complex‐mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty‐seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2‐dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABAA receptor (GABAAR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABAAR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABAAR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABAAR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABAAR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABAAR α3 interacts with gephyrin, GABAAR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABAAR α2 and collybistin or GABAAR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABAAR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABAAR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABAAR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABAAR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABAARs, but not GlyRs or other GABAAR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABAARs containing the α2 subunit.

Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of γ-aminobutyric acid A (GABAA) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2–3 consecutive days for completion.

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat.

The tubulin‐binding protein gephyrin is essential for the formation of postsynaptic glycine‐receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of γ‐aminobutyric acid (GABA) and of two major GABAA‐receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the α1 and γ2 subunits of the GABAA receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABAA‐receptor subtypes in the postsynaptic membrane. J. Comp. Neurol. 395:231–244, 1998.