RESUMEN
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.
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Receptores AMPA , Sinapsis , Receptores AMPA/metabolismo , Receptores AMPA/química , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Humanos , Transmisión Sináptica/fisiología , Nanoestructuras/químicaRESUMEN
Neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism, involve altered synaptic transmission and plasticity. Functional characterization of their associated genes is vital for understanding physio-pathological brain functions. LGI3 is a recently recognized ID-associated gene encoding a secretory protein related to an epilepsy-gene product, LGI1. Here, we find that LGI3 is uniquely secreted from oligodendrocytes in the brain and enriched at juxtaparanodes of myelinated axons, forming nanoscale subclusters. Proteomic analysis using epitope-tagged Lgi3 knockin mice shows that LGI3 uses ADAM23 as a receptor and selectively co-assembles with Kv1 channels. A lack of Lgi3 in mice disrupts juxtaparanodal clustering of ADAM23 and Kv1 channels and suppresses Kv1-channel-mediated short-term synaptic plasticity. Collectively, this study identifies an extracellular organizer of juxtaparanodal Kv1 channel clustering for finely tuned synaptic transmission. Given the defective secretion of the LGI3 missense variant, we propose a molecular pathway, the juxtaparanodal LGI3-ADAM23-Kv1 channel, for understanding neurodevelopmental disorders.
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Epilepsia , Proteómica , Animales , Ratones , Axones/metabolismo , Epilepsia/metabolismo , Plasticidad Neuronal , Oligodendroglía/metabolismo , Proteínas/metabolismoRESUMEN
Pathogenic variants of ADAM22 affecting either its biosynthesis and/or its interactions with either LGI1 and/or PSD-95 have been recently identified in individuals with developmental and epileptic encephalopathy. Here, we describe a girl with seizures, delayed psychomotor development, and behavioural disorder, carrying a homozygous variant in ADAM22 (NM_021723.5:c.2714C > T). The variant has a surprisingly high frequency in the Roma population of the Czech and Slovak Republic, with 11 of 213 (â¼5.2%) healthy Roma individuals identified as heterozygous carriers. Structural in silico characterization revealed that the genetic variant encodes the missense variant p.S905F, which localizes to the PDZ-binding motif of ADAM22. Studies in transiently transfected mammalian cells revealed that the variant has no effect on biosynthesis and stability of ADAM22. Rather, protein-protein interaction studies showed that the p.S905F variant specifically impairs ADAM22 binding to PSD-95 and other proteins from a family of membrane-associated guanylate kinases, while it has only minor effect on ADAM22-LGI1 interaction. Our study indicates that a significant proportion of epilepsy in patients of Roma ancestry may be caused by homozygous c.2714C > T variants in ADAM22. The study of this ADAM22 variant highlights a novel pathogenic mechanism of ADAM22 dysfunction and reconfirms an essential role of interaction of ADAM22 with membrane-associated guanylate kinases in seizure protection in humans.
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BACKGROUND AND OBJECTIVES: The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. RESULTS: Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. DISCUSSION: IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.
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Autoanticuerpos , Síndrome de Guillain-Barré , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante , Anciano , Animales , Humanos , Ratones , Persona de Mediana Edad , Ratas , Proteínas ADAM , Autoanticuerpos/sangre , Autoanticuerpos/química , Síndrome de Guillain-Barré/diagnóstico , Células HEK293 , Inmunoglobulina G , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/inmunología , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/patologíaRESUMEN
Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.
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Cisteína , Monofenol Monooxigenasa , Humanos , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Lipoilación , Aciltransferasas/metabolismo , Melaninas/metabolismo , Melanocitos/metabolismoRESUMEN
The small GTPase Cdc42 exists in the form of two alternatively spliced variants that are modified by hydrophobic chains: the ubiquitously expressed Cdc42-prenyl and a brain-specific isoform that can be palmitoylated, Cdc42-palm. Our previous work demonstrated that Cdc42-palm can be palmitoylated at two cysteine residues, Cys188 and Cys189, while Cys188 can also be prenylated. We showed that palmitoylation of Cys188 is essential for the plasma membrane localization of Cdc42-palm and is critically involved in Cdc42-mediated regulation of gene transcription and neuronal morphology. However, the abundance and regulation of this modification was not investigated. In the present study, we found that only a minor fraction of Cdc42 undergoes monopalmitoylation in neuroblastoma cells and in hippocampal neurons. In addition, we identified DHHC5 as one of the major palmitoyl acyltransferases that could physically interact with Cdc42-palm. We demonstrate that overexpression of dominant negative DHHC5 mutant decreased palmitoylation and plasma membrane localization of Cdc42-palm. In addition, knockdown of DHHC5 significantly reduced Cdc42-palm palmitoylation, leading to a decrease of Cdc42-mediated gene transcription and spine formation in hippocampal neurons. We also found that the expression of DHHC5 in the brain is developmentally regulated. Taken together, these findings suggest that DHHC5-mediated palmitoylation of Cdc42 represents an important mechanism for the regulation of Cdc42 functions in hippocampus.
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Aciltransferasas , Lipoilación , Proteínas de la Membrana , Proteínas de Unión al GTP Monoméricas , Neuronas , Columna Vertebral , Proteína de Unión al GTP cdc42 , Aciltransferasas/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuronas/citología , Columna Vertebral/crecimiento & desarrollo , Transcripción Genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
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.
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Cerebelo , Canales Iónicos , Animales , Canales Iónicos/metabolismo , RatonesRESUMEN
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.
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Proteínas ADAM , Encefalopatías , Epilepsia Refractaria , Proteínas del Tejido Nervioso , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Atrofia , Encefalopatías/genética , Homólogo 4 de la Proteína Discs Large , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed 'R-GenGAR-DA'. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = - 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.
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Técnicas Biosensibles , Dopamina , Animales , Dopamina/metabolismo , Células HeLa , Humanos , Neuronas/metabolismo , Norepinefrina/metabolismo , Norepinefrina/farmacologíaRESUMEN
What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that â¼50% of LGI1 and â¼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment.
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Proteínas 14-3-3/metabolismo , Proteínas ADAM/fisiología , Encéfalo/metabolismo , Epilepsia/prevención & control , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Proteínas del Tejido Nervioso/fisiología , Proteínas 14-3-3/genética , Animales , Encéfalo/patología , Epilepsia/metabolismo , Epilepsia/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition. mAbs selectively reduced GABAergic currents in neuronal cultures without causing receptor internalization. Cerebroventricular infusion of GABAAR mAbs and Fab fragments into rodents induced a severe phenotype with seizures and increased mortality, reminiscent of encephalitis patients' symptoms. Our results demonstrate direct pathogenicity of autoantibodies on GABAARs independent of Fc-mediated effector functions and provide an animal model for GABAAR encephalitis. They further provide the scientific rationale for clinical treatments using antibody depletion and can serve as tools for the development of antibody-selective immunotherapies.
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Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Encefalitis/inmunología , Epilepsia/inmunología , Receptores de GABA-A/inmunología , Convulsiones/inmunología , Animales , Autoantígenos/inmunología , Células Cultivadas , Células HEK293 , Hipocampo/inmunología , Humanos , Ratones , Neuronas/inmunologíaRESUMEN
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.
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Guanilato-Quinasas/metabolismo , Potenciación a Largo Plazo/fisiología , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Transporte de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Exquisitely-regulated synaptic transmission and plasticity underlie higher brain functions such as learning and memory. PSD-95, a member of the MAGUK family, scaffolds an array of postsynaptic proteins including AMPA and NMDA receptors, and plays essential roles in excitatory synaptic transmission and postsynaptic organization. Epilepsy-related secreted protein LGI1 and its receptor ADAM22 represent major constituent elements of the PSD-95-containing synaptic protein complex in the brain. Recent studies begin to reveal a trans-synaptic configuration of the LGI1-ADAM22 complex and its pivotal role in AMPA and NMDA receptor-mediated synaptic transmission through regulating MAGUKs. Especially interesting is that without the association with LGI1-ADAM22, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Here, we review roles of LGI1-ADAM22 in synaptic function, and discuss its modes of action on the MAGUK regulation: as (i) a trans-synaptic hub, (ii) an extracellular scaffold, and (iii) an allosteric activator. We also highlight patho-physiological roles of the LGI1-ADAM22-MAGUK linkage in synaptic disorders such as epilepsy and autoimmune limbic encephalitis.
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Proteínas ADAM/metabolismo , Guanilato-Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Encéfalo/metabolismo , Epilepsia/metabolismo , Humanos , Transmisión SinápticaRESUMEN
Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.
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Trastorno del Espectro Autista/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Secuencia de Aminoácidos , Animales , Trastorno del Espectro Autista/metabolismo , Escala de Evaluación de la Conducta , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Dominios Proteicos , Empalme de Proteína , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Recombinantes , Transducción de Señal/genética , Transducción de Señal/fisiología , Conducta Social , Sinapsis/genéticaRESUMEN
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.
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Proteínas ADAM/genética , Epilepsia/genética , Guanilato-Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al Calcio/genética , Modelos Animales de Enfermedad , Epilepsia/patología , Epilepsia/prevención & control , Técnicas de Sustitución del Gen , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Proteínas de la Membrana/genética , Ratones , Moléculas de Adhesión de Célula Nerviosa/genética , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Canales de Potasio de la Superfamilia Shaker/genéticaRESUMEN
Optogenetics has become an indispensable tool for investigating brain functions. Although non-human primates are particularly useful models for understanding the functions and dysfunctions of the human brain, application of optogenetics to non-human primates is still limited. In the present study, we generate an effective adeno-associated viral vector serotype DJ to express channelrhodopsin-2 (ChR2) under the control of a strong ubiquitous CAG promoter and inject into the somatotopically identified forelimb region of the primary motor cortex in macaque monkeys. ChR2 is strongly expressed around the injection sites, and optogenetic intracortical microstimulation (oICMS) through a homemade optrode induces prominent cortical activity: Even single-pulse, short-duration oICMS evokes long-lasting repetitive firings of cortical neurons. In addition, oICMS elicits distinct forelimb movements and muscle activity, which are comparable to those elicited by conventional electrical ICMS. The present study removes obstacles to optogenetic manipulation of neuronal activity and behaviors in non-human primates.
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Miembro Anterior/fisiología , Corteza Motora/fisiología , Movimiento/fisiología , Optogenética , Animales , Channelrhodopsins/metabolismo , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Macaca , Neuronas/fisiología , Estimulación FísicaRESUMEN
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.
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Colesterol/genética , Lipoilación/genética , Microdominios de Membrana/genética , Receptores de Lipoproteína/genética , Comunicación Celular/genética , Colesterol/metabolismo , Cisteína/química , Cisteína/genética , Células Epiteliales/metabolismo , Humanos , Uniones Intercelulares/genética , Proteína 2 con Dominio MARVEL , Microdominios de Membrana/química , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , Receptores de Lipoproteína/química , Uniones Estrechas/genética , Uniones Estrechas/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Autoanticuerpos/farmacología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neuronas/fisiología , Proteínas ADAM/efectos de los fármacos , Anciano , Animales , Anticuerpos Monoclonales/líquido cefalorraquídeo , Autoanticuerpos/líquido cefalorraquídeo , Región CA3 Hipocampal/fisiología , Células Cultivadas , Encefalitis/líquido cefalorraquídeo , Encefalitis/inmunología , Femenino , Enfermedad de Hashimoto/líquido cefalorraquídeo , Enfermedad de Hashimoto/inmunología , Humanos , Isotipos de Inmunoglobulinas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteínas del Tejido Nervioso/efectos de los fármacos , Ratas , Transmisión Sináptica/efectos de los fármacosRESUMEN
Dickkopf1 (DKK1) was originally identified as an antagonist of Wnt signaling that binds to and induces the clathrin-mediated endocytosis of the Wnt coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). DKK1 also binds to cytoskeleton-associated protein 4 (CKAP4), which was originally identified as an endoplasmic reticulum (ER) protein but also functions at the plasma membrane as a receptor for various ligands. The DKK1-CKAP4 pathway is activated in several human cancers and promotes cell proliferation by activating signaling through the kinases PI3K and AKT. We found that both CKAP4 and LRP6 primarily localized to detergent-resistant membrane (DRM) fractions of the plasma membrane in a palmitoylation-dependent manner and that palmitoylation of CKAP4 was required for it to promote cell proliferation. DKK1 induced the depalmitoylation of both CKAP4 and LRP6 by acylprotein thioesterases (APTs), resulting in their translocation to the non-DRM fractions. Moreover, DKK1-dependent depalmitoylation of both receptors required activation of the PI3K-AKT pathway. DKK1 simultaneously bound CKAP4 and LRP6, resulting in the formation of a ternary complex. LRP5/6 knockdown decreased DKK1-dependent AKT activation and cancer cell proliferation through CKAP4, whereas CKAP4 knockdown did not affect DKK1-dependent inhibition of Wnt signaling through LRP5/6. These results indicate that the palmitoylation states of CKAP4 and LRP6 play important roles in their signaling and that LRP5/6 enhance DKK1-CKAP4 signaling.