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.
Asunto(s)
Receptores AMPA , Sinapsis , Receptores AMPA/metabolismo , Receptores AMPA/química , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Humanos , Transmisión Sináptica/fisiología , Nanoestructuras/químicaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
Cerebelo , Canales Iónicos , Animales , Canales Iónicos/metabolismo , RatonesRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Esterasas/fisiología , Homeostasis , Peroxirredoxinas/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismo , Oxidación-ReducciónRESUMEN
The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.
Asunto(s)
Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Unión Proteica , Proteína ReelinaRESUMEN
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.
Asunto(s)
Canales de Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteómica , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Canales de Calcio/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Mapeo de Interacción de Proteínas/métodos , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Proteínas ADAM/metabolismo , Regulación de la Expresión Génica , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Sinapsis/fisiología , Secuencias de Aminoácidos , Animales , Encéfalo/patología , Membrana Celular/metabolismo , Homólogo 4 de la Proteína Discs Large , Electrodos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Neuronas/metabolismo , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transmisión SinápticaRESUMEN
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.
Asunto(s)
Guanilato-Quinasas/metabolismo , Lipoilación/fisiología , Proteínas de la Membrana/metabolismo , Monoacilglicerol Lipasas/metabolismo , Neuronas/enzimología , Serina/análogos & derivados , Animales , Línea Celular Transformada , Cerebelo/metabolismo , Chlorocebus aethiops , Homólogo 4 de la Proteína Discs Large , Femenino , Guanilato-Quinasas/genética , Hipocampo/citología , Hidrolasas/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Monoacilglicerol Lipasas/genética , Palmitatos/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Serina/aislamiento & purificación , Serina/metabolismo , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato , Tritio/metabolismoRESUMEN
Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile-onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte-specific Mlc1 overexpression causes infantile-onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+ /K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+ /K+ pump. In contrast, no changes in Na+ /K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+ /K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile-onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile-onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150-168.
Asunto(s)
Astrocitos/metabolismo , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/genética , Sustancia Blanca/metabolismo , Animales , Membrana Celular/metabolismo , Quistes/genética , Modelos Animales de Enfermedad , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Mutación/genéticaRESUMEN
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.
Asunto(s)
Glicoproteínas/química , Glicoproteínas/metabolismo , Vaina de Mielina/metabolismo , Filogenia , Proteínas ADAM/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Axones/metabolismo , Secuencia Conservada , Prueba de Complementación Genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Sistema Nervioso Periférico/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Relación Estructura-Actividad , Pez CebraRESUMEN
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.
Asunto(s)
Lipoilación , Microdominios de Membrana/metabolismo , Animales , Humanos , Espacio Intracelular/metabolismo , Proteínas/metabolismoRESUMEN
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.
Asunto(s)
Autoanticuerpos/sangre , Encefalopatías/sangre , Encefalopatías/inmunología , Encéfalo/patología , Enfermedad de Hashimoto/sangre , Enfermedad de Hashimoto/inmunología , Receptores de GABA-A/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/inmunología , Encéfalo/metabolismo , Encefalopatías/complicaciones , Encefalopatías/patología , Células Cultivadas , Chlorocebus aethiops , Trastornos del Conocimiento/etiología , Encefalitis , Femenino , Enfermedad de Hashimoto/complicaciones , Enfermedad de Hashimoto/patología , Hipocampo/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteínas/inmunología , RatasRESUMEN
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.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoilación/genética , Proteínas de la Membrana/metabolismo , Transmisión Sináptica/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Homólogo 4 de la Proteína Discs Large , Hipocampo/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neuronas/metabolismo , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
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.
Asunto(s)
Lipoilación/fisiología , Neurogénesis/fisiología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Microdominios de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transporte de ProteínasRESUMEN
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.