Celebrating the Birthday of AMPA Receptor Nanodomains: Illuminating the Nanoscale Organization of Excitatory Synapses with 10 Nanocandles.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology.

MAGUKs are essential, but redundant, in long-term potentiation.

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention.

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Biallelic ADAM22 pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy.

Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts.

Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.

Human Cerebrospinal Fluid Monoclonal LGI1 Autoantibodies Increase Neuronal Excitability.

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. INTERPRETATION: Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.

ABHD10 is an S-depalmitoylase affecting redox homeostasis through peroxiredoxin-5.

S-Palmitoylation is a reversible lipid post-translational modification that has been observed on mitochondrial proteins, but both the regulation and functional consequences of mitochondrial S-palmitoylation are poorly understood. Here, we show that perturbing the 'erasers' of S-palmitoylation, acyl protein thioesterases (APTs), with either pan-active inhibitors or a mitochondrial-targeted APT inhibitor, diminishes the antioxidant buffering capacity of mitochondria. Surprisingly, this effect was not mediated by the only known mitochondrial APT, but rather by a resident mitochondrial protein with no known endogenous function, ABHD10. We show that ABHD10 is a member of the APT family of regulatory proteins and identify peroxiredoxin-5 (PRDX5), a key antioxidant protein, as a target of ABHD10 S-depalmitoylase activity. We then find that ABHD10 regulates the S-palmitoylation status of the nucleophilic active site residue of PRDX5, providing a direct mechanistic connection between ABHD10-mediated S-depalmitoylation of PRDX5 and its antioxidant capacity.

Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

Coupling of a voltage-gated Ca2+ channel homologue with a plasma membrane H+ -ATPase in yeast.

Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+ . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α1 and auxiliary α2 /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+ -ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+ -pumping activity.

The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function.

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

Identification of PSD-95 Depalmitoylating Enzymes.

UNLABELLED: Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/ß-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT: Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.

Ivermectin activates GIRK channels in a PIP2 -dependent, Gßγ -independent manner and an amino acid residue at the slide helix governs the activation.

KEY POINTS: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. We found that the G-protein-gated inwardly rectifying K+ (GIRK) channel, especially GIRK2, is activated by IVM directly in a Gßγ -independent manner, but the activation is dependent on phosphatidylinositol-4,5-biphosphate (PIP2 ). We identified a critical amino acid residue of GIRK2 for activation by IVM, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD). The results demonstrate that the TM-CTD interface in GIRK channel, rather than the TMs, governs IVM-mediated activation and provide us with novel insights on the mode of action of IVM in ion channels. ABSTRACT: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. In this study, we found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. Electrophysiological recordings in Xenopus oocytes revealed that IVM activates GIRK channel in a phosphatidylinositol-4,5-biphosphate (PIP2 )-dependent manner, and that the IVM-mediated GIRK activation is independent of Gßγ subunits. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. Chimeric and mutagenesis analyses identified an amino acid residue unique to GIRK2 among the GIRK family, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM-CTD interface in GIRK channels, rather than the TMs, governs IVM-mediated activation. These findings provide us with novel insights on the mode of action of IVM in ion channels that could lead to identification of new pharmacophores which activate the GIRK channel.

Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination.

The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.

Local Palmitoylation Cycles and Specialized Membrane Domain Organization.

Palmitoylation is an evolutionally conserved lipid modification of proteins. Dynamic and reversible palmitoylation controls a wide range of molecular and cellular properties of proteins including the protein trafficking, protein function, protein stability, and specialized membrane domain organization. However, technical difficulties in (1) detection of palmitoylated substrate proteins and (2) purification and enzymology of palmitoylating enzymes have prevented the progress in palmitoylation research, compared with that in phosphorylation research. The recent development of proteomic and chemical biology techniques has unexpectedly expanded the known complement of palmitoylated proteins in various species and tissues/cells, and revealed the unique occurrence of palmitoylated proteins in membrane-bound organelles and specific membrane compartments. Furthermore, identification and characterization of DHHC (Asp-His-His-Cys) palmitoylating enzyme-substrate pairs have contributed to elucidating the regulatory mechanisms and pathophysiological significance of protein palmitoylation. Here, we review the recent progress in protein palmitoylation at the molecular, cellular, and in vivo level and discuss how locally regulated palmitoylation machinery works for dynamic nanoscale organization of membrane domains.

Identification and characterization of GABA(A) receptor autoantibodies in autoimmune encephalitis.

Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the ß3 subunit of the GABA(A) receptor. The ß3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the ß3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies.

Postsynaptic nanodomains generated by local palmitoylation cycles.

Precise regulation of protein assembly at specialized membrane domains is essential for diverse cellular functions including synaptic transmission. However, it is incompletely understood how protein clustering at the plasma membrane is initiated, maintained and controlled. Protein palmitoylation, a common post-translational modification, regulates protein targeting to the plasma membrane. Such modified proteins are enriched in these specialized membrane domains. In this review, we focus on palmitoylation of PSD-95, which is a major postsynaptic scaffolding protein and makes discrete postsynaptic nanodomains in a palmitoylation-dependent manner and discuss a determinant role of local palmitoylation cycles in creating highly localized hotspots at the membrane where specific proteins concentrate to organize functional domains.

Protein palmitoylation in neuronal development and synaptic plasticity.

Protein palmitoylation, a classical and common lipid modification, regulates diverse aspects of neuronal protein trafficking and function. The reversible nature of palmitoylation provides a potential general mechanism for protein shuttling between intracellular compartments. The recent discovery of palmitoylating enzymes--a large DHHC (Asp-His-His-Cys) protein family--and the development of new proteomic and imaging methods have accelerated palmitoylation analysis. It is becoming clear that individual DHHC enzymes generate and maintain the specialized compartmentalization of substrates in polarized neurons. Here, we discuss the regulatory mechanisms for dynamic protein palmitoylation and the emerging roles of protein palmitoylation in various aspects of pathophysiology, including neuronal development and synaptic plasticity.

Autoantibodies to epilepsy-related LGI1 in limbic encephalitis neutralize LGI1-ADAM22 interaction and reduce synaptic AMPA receptors.

More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures. We comprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain of ADAM22 was sufficient to reduce synaptic AMPA receptors in rat hippocampal neurons and that levels of AMPA receptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces the AMPA receptor function, causing epileptic disorders. These results suggest that by finely regulating the synaptic AMPA receptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life.

In silico screening for palmitoyl substrates reveals a role for DHHC1/3/10 (zDHHC1/3/11)-mediated neurochondrin palmitoylation in its targeting to Rab5-positive endosomes.

Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca(2+)/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.