Gephyrin palmitoylation and oligomerization in synaptic plasticity, membrane recruitment and proteostasis

Efficient signal transmission in the central nervous system is essential for higher brain functions. Inhibitory signaling in the brain primarily takes place at GABAergic (γ-aminobutyric acid) synapses and balances the activity of excitatory synapses. GABA type A receptors (GABAARs) are clustered at the synapse by a scaffold with the peripheral membrane protein gephyrin as major postsynaptic protein. One key feature of synapses is their ability to adapt in response to neuronal network activities, though underlying mechanisms how these changes are orchestrated under normal and pathological conditions are poorly understood. The first part of the study focuses on gephyrin’s membrane association in neurons with the aim to elucidate the molecular mechanisms and significance of membrane tethering. Gephyrin from Sf9 insect cells, but not E. coli, bound to liposomes and specifically to phosphatidylinositol 4-phosphate in protein-lipid overlay assays. Furthermore, gephyrin was identified to be mainly associated with cholesterol-rich membrane microdomains in mouse brains. Posttranslational lipid modifications of synaptic proteins regulate their trafficking and membrane localization and contribute to synaptic plasticity. Using different experimental approaches, gephyrin was identified to be palmitoylated in vivo. Palmitoylation of gephyrin was crucial for its localization at synapses and influenced the size of gephyrin clusters and the architecture of the inhibitory synapse. Membrane release of gephyrin upon inhibition of palmitoylation led to reduced surface quantities of synaptic GABAAR subunits. Additionally, the membrane detachment made gephyrin more susceptible to cleavage by the protease calpain I resulting in an accelerated turnover of the protein. Gephyrin palmitoylation was identified to be regulated by GABAAR activity leading to rapid changes in gephyrin palmitoylation levels. Palmitoylation screens in hippocampal neurons identified the neurite-localized palmitoyl transferase DHHC12 and Golgi-resident DHHC16 as gephyrin palmitoylating enzymes, which increase gephyrin cluster size and its amount in synapses. Gephyrin was also identified to be physiologically S-nitrosylated by NO, which is produced by neuronal nitric oxide synthase (nNOS). Pharmacological nNOS activity modulation in HEK-293 cells and hippocampal neurons reciprocally regulated gephyrin palmitoylation. Together, these findings identify differential modification of gephyrin by palmitoylation and nitrosylation and suggest that palmitoylation dynamics of gephyrin contribute to the regulation of GABAergic activity-dependent plasticity. Inhibitory signaling is crucial to counterbalance excitatory transmission. Anomalous inhibitory circuits and particularly irregular gephyrin expression have been linked to epileptic disorders. Patients with idiopathic generalized epilepsy have been screened for mutations in the GPHN gene. In the second part, a patient was identified with a hemizygous mutation in GPHN resulting in the expression of a truncated gephyrin variant that failed to oligomerize at inhibitory synapses. This pathogenic variant acted dominant-negatively on regular gephyrin and disrupted the normal gephyrin scaffold and synaptic GABAAR clustering in hippocampal neurons. The results suggest that mutations in genes coding for proteins of the inhibitory synapse is an important mechanism in the pathophysiology of monogenetic epilepsy forms

Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy

Correction to: EMBO Mol Med (2015) 7: 1580–1594. DOI 10.15252/emmm.201505323 | Published online 27 November 2015 EMBO Molecular Medicine 2017 vol 9 No12: 1764.Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition plays an important role in neurological disorders. Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABA(A) and glycine receptors (GABA(A)Rs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. Additionally, gephyrin catalyzes the synthesis of the molybdenum cofactor (MoCo) in peripheral tissue. We identified a de novo missense mutation (G375D) in the gephyrin gene (GPHN) in a patient with epileptic encephalopathy resembling Dravet syndrome. Although stably expressed and correctly folded, gephyrin-G375D was non-synaptically localized in neurons and acted dominant-negatively on the clustering of wild- type gephyrin leading to a marked decrease in GABA(A)R surface expression and GABAergic signaling. We identified a decreased binding affinity between gephyrin-G375D and the receptors, suggesting that Gly375 is essential for gephyrin-receptor complex formation. Surprisingly, gephyrin-G375D was also unable to synthesize MoCo and activate MoCo-dependent enzymes. Thus, we describe a missense mutation that affects both functions of gephyrin and suggest that the identified defect at GABAergic synapses is the mechanism underlying the patient's severe phenotype.Peer reviewe

Evaluation of presumably disease causing SCN1A variants in a cohort of common epilepsy syndromes

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation: We identified 8 known missense mutations, previously reported as path

Neuronal Nitric Oxide Synthase-Dependent S-Nitrosylation of Gephyrin Regulates Gephyrin Clustering at GABAergic Synapses

Gephyrin, the principal scaffolding protein at inhibitory synapses, is essential for postsynaptic clustering of glycine and GABA type A receptors (GABA(A)Rs). Gephyrin cluster formation, which determines the strength of GABAergic transmission, is modulated by interaction with signaling proteins and post-translational modifications. Here, we show that gephyrin was found to be associated with neuronal nitric oxide synthase (nNOS), the major source of the ubiquitous and important signaling molecule NO in brain. Furthermore, we identified that gephyrin is S-nitrosylated in vivo. Overexpression of nNOS decreased the size of postsynaptic gephyrin clusters in primary hippocampal neurons. Conversely, inhibition of nNOS resulted in a loss of S-nitrosylation of gephyrin and the formation of larger gephyrin clusters at synaptic sites, ultimately increasing the number of cell surface expressed synaptic GABA(A)Rs. In conclusion, S-nitrosylation of gephyrin is important for homeostatic assembly and plasticity of GABAergic synapses

Deep proteomics identifies shared molecular pathway alterations in synapses of patients with schizophrenia and bipolar disorder and mouse model

Summary: Synaptic dysfunction is implicated in the pathophysiology of schizophrenia (SCZ) and bipolar disorder (BP). We use quantitative mass spectrometry to carry out deep, unbiased proteomic profiling of synapses purified from the dorsolateral prefrontal cortex of 35 cases of SCZ, 35 cases of BP, and 35 controls. Compared with controls, SCZ and BP synapses show substantial and similar proteomic alterations. Network analyses reveal upregulation of proteins associated with autophagy and certain vesicle transport pathways and downregulation of proteins related to synaptic, mitochondrial, and ribosomal function in the synapses of individuals with SCZ or BP. Some of the same pathways are similarly dysregulated in the synaptic proteome of mutant mice deficient in Akap11, a recently discovered shared risk gene for SCZ and BP. Our work provides biological insights into molecular dysfunction at the synapse in SCZ and BP and serves as a resource for understanding the pathophysiology of these disorders

Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses

Postsynaptic scaffolding proteins regulate coordinated neurotransmission by anchoring and clustering receptors and adhesion molecules. Gephyrin is the major instructive molecule at inhibitory synapses, where it clusters glycine as well as major subsets of GABA type A receptors (GABA(A)Rs). Here, we identified palmitoylation of gephyrin as an important mechanism of strengthening GABAergic synaptic transmission, which is regulated by GABA(A)R activity. We mapped palmitoylation to Cys212 and Cys284, which are critical for both association of gephyrin with the postsynaptic membrane and gephyrin clustering. We identified DHHC-12 as the principal palmitoyl acyltransferase that palmitoylates gephyrin. Furthermore, gephyrin pamitoylation potentiated GABAergic synaptic transmission, as evidenced by an increased amplitude of miniature inhibitory postsynaptic currents. Consistently, inhibiting gephyrin palmitoylation either pharmacologically or by expression of palmitoylation-deficient gephyrin reduced the gephyrin cluster size. In aggregate, our study reveals that palmitoylation of gephyrin by DHHC-12 contributes to dynamic and functional modulation of GABAergic synapses

Model for palmitoylation-mediated regulation of gephyrin clustering and plasticity.

<p>Gephyrin, palmitoylated on Cys212 and 284, forms stable clusters at the postsynaptic membrane. Activity of GABAergic synapses promotes (de)palmitoylation of gephyrin by yet unknown mechanisms. Silencing of GABAergic transmission leads to gephyrin depalmitoylation and membrane release, ultimately decreasing the size of gephyrin clusters. The palmitoyl transferase DHHC-12, localized in the Golgi in neurons including presumed Golgi outposts in primary dendritic shafts, is the principle gephyrin-palmitoylating enzyme and allows dynamic (re)palmitoylation of gephyrin.</p

Palmitoylation on Cys212 and Cys284 is essential for gephyrin localization and clustering in neurons.

<p>(a) Scheme of gephyrin domain architecture with surface-exposed and Palm-CSS 3.0–predicted cysteine residues. (b) Immunoprecipitation of ABE-assay–processed gephyrin cysteine-to-serine mutants expressed in HEK293 cells. Streptavidin-HRP shows palmitoylation status of individual variants, whereas GFP-immunoblot was used as the loading control. Experiment was repeated three times with similar results. (c) SDS-PAGE of ABE-assay–processed and Ni-NTA affinity-purified gephyrin and LC-MS/MS analysis of the protein bands. (d) Quantitative comparison of cluster size of GFP-tagged WT and mutant gephyrin variants in primary hippocampal cultures. (e) Total intensity of WT gephyrin or 212,284Geph clusters (f) Representative dendrites of gephyrin-GFP and 212,284Geph. Scale bar, 5 µm. All quantifications are means ± SEM (***<i>p</i><0.001 using Student's <i>t</i> test; NS, not significant). At least three independent cultures were used per experiment.</p

Gephyrin is palmitoylated by DHHC-12.

<p>(a) Individual HA-tagged DHHC proteins or GST-HA as control were co-expressed with gephyrin-GFP for 24 h in HEK293 cells and analyzed with the ABE assay. Omitting hydroxylamine demonstrated specify of the assay. (b) Gephyrin-GFP was co-expressed with individual HA-tagged DHHC enzymes or GST-HA as control in primary hippocampal neurons, and gephyrin cluster size was quantified by confocal laser scanning microscopy. In the histogram, DHHC enzymes are labeled according to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001908#pbio.1001908-Fukata3" target="_blank">[16]</a>. (c) Representative images show phenotypical differences of gephyrin-GFP clusters in DHHC-12–expressing neurons compared to GST-expressing controls. Scale bar, 10 µm. (d) Gephyrin-GFP cluster density after 24 h expression of DHHC-12 compared to control neurons. (e–g) Quantification of gephyrin-GFP cluster size and density and representative image upon expression of dominant-negative DHH<u>S</u>-12–HA compared to mCherry-expressing control neurons. Scale bar, 5 µm. (h) Quantitative analysis of 212,284Geph cluster size in control mCherry and DHHC-12-HA–expressing neurons. (i) Expression of <i>dhhc12</i> mRNA in cultured hippocampal neurons (h. neurons) was validated by PCR using cDNA prepared from neurons. A plasmid encoding the respective <i>dhhc</i> gene was used as control. (j) Primary hippocampal neurons were grown in medium containing cell-penetrating siRNAs to knock down expression of DHHC-12 or DHHC-16. Lysates were subjected to ABE, and the palmitoylation level of gephyrin, GABA_AR γ2, and PSD-95 was analyzed by immunoblotting. We used 10% of the lysate to detect total levels of the respective proteins; scr., scrambled. (k) Quantification of three independent experiments shows gephyrin palmitoylation levels normalized to total protein (***<i>p</i><0.001 using Student's <i>t</i> test).</p