RESUMO
A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsynytenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, p < 0.04), a finding that was replicated (0.7 to 0.8-fold, p < 0.05) for 6 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Proteômica/métodos , Sinapses/metabolismo , Idoso , Doença de Alzheimer/metabolismo , Autopsia , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/líquido cefalorraquidiano , Proteínas de Ligação ao Cálcio/metabolismo , Diagnóstico Precoce , Feminino , Humanos , Masculino , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/metabolismo , Sintomas Prodrômicos , Prognóstico , Receptores de AMPA/metabolismo , Sintaxina 1/líquido cefalorraquidiano , Sintaxina 1/metabolismo , Antígenos Thy-1/líquido cefalorraquidiano , Antígenos Thy-1/metabolismoRESUMO
The SynGAP protein is a major regulator of synapse biology and neural circuit function. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic function and neural excitability. SynGAP has been involved in multiple signaling pathways and can regulate small GTPases with very different roles. Yet, the molecular bases behind this pleiotropy are poorly understood. We hypothesize that different SynGAP isoforms will mediate different sets of functions and that deciphering their spatio-temporal expression and subcellular localization will accelerate understanding their multiple functions. Using isoform-specific antibodies recognizing SynGAP in mouse and human samples we found distinctive developmental expression patterns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic density-95, in cortex and hippocampus during the first 2 weeks of postnatal development. Suggesting that during this period other isoforms would have a more prominent role. Furthermore, we observed subcellular localization differences between isoforms, particularly throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, SynGAP was predominantly found in non-synaptic locations in a period of early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms were always found enriched in the postsynaptic density, α2 isoforms changed from a non-synaptic to a mostly postsynaptic density localization with age and ß isoforms were always found enriched in non-synaptic locations. The differential expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.
Assuntos
Encéfalo/crescimento & desenvolvimento , Proteínas Ativadoras de ras GTPase/genética , Animais , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Simulação por Computador , Regulação da Expressão Gênica no Desenvolvimento/genética , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Humanos , Isomerismo , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteômica , Frações Subcelulares/metabolismo , Proteínas Ativadoras de ras GTPase/biossínteseRESUMO
Neurotransmitter diseases are a well-defined group of metabolic conditions caused, in most instances, by genes specifically expressed in the presynaptic button. Better understanding of presynaptic molecular physiology, both in normal and pathological conditions, should help develop therapeutical strategies. The clinical relevance of the presynapse in inherited metabolic disorders is in glaring contrast with that of the postsynaptic component, which so far does not seem to play a relevant role in these disorders. This is somewhat surprising, as postsynaptic proteins are known to be involved in many nervous system diseases, particularly in neurodevelopmental and psychiatric disorders. The goal of this article is to explore if defects in the sophisticated postsynaptic machinery could also have a role in neurometabolic disorders.
Assuntos
Doenças Metabólicas/genética , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Proteoma , Humanos , Doenças Metabólicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Sinapses/metabolismo , Sinapses/patologiaRESUMO
Advances in technology have equipped the field of neuroproteomics with refined tools for the study of the expression, interaction and function of proteins in the nervous system. In combination with bioinformatics, neuroproteomics can address the organization of dynamic, functional protein networks and macromolecular structures that underlie physiological, anatomical and behavioural processes. Furthermore, neuroproteomics is contributing to the elucidation of disease mechanisms and is a powerful tool for the identification of biomarkers.
Assuntos
Encéfalo/fisiologia , Proteômica/métodos , Animais , Biomarcadores/análise , Humanos , Rede Nervosa/fisiologia , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Proteômica/tendênciasRESUMO
SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials.
RESUMO
Understanding the origins and evolution of synapses may provide insight into species diversity and the organization of the brain. Using comparative proteomics and genomics, we examined the evolution of the postsynaptic density (PSD) and membrane-associated guanylate kinase (MAGUK)-associated signaling complexes (MASCs) that underlie learning and memory. PSD and MASC orthologs found in yeast carry out basic cellular functions to regulate protein synthesis and structural plasticity. We observed marked changes in signaling complexity at the yeast-metazoan and invertebrate-vertebrate boundaries, with an expansion of key synaptic components, notably receptors, adhesion/cytoskeletal proteins and scaffold proteins. A proteomic comparison of Drosophila and mouse MASCs revealed species-specific adaptation with greater signaling complexity in mouse. Although synaptic components were conserved amongst diverse vertebrate species, mapping mRNA and protein expression in the mouse brain showed that vertebrate-specific components preferentially contributed to differences between brain regions. We propose that the evolution of synapse complexity around a core proto-synapse has contributed to invertebrate-vertebrate differences and to brain specialization.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Estudos de Avaliação como Assunto , Proteínas do Tecido Nervoso/metabolismo , Proteoma , Sinapses/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Comportamento Animal , Encéfalo/citologia , Encéfalo/metabolismo , Mapeamento Encefálico , Proteínas Adaptadoras de Sinalização CARD , Proteínas do Citoesqueleto/genética , Drosophila , Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteômica/métodos , Transdução de Sinais/fisiologiaRESUMO
Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.
Assuntos
Convulsões , Proteínas Ativadoras de ras GTPase , Animais , Cognição , Camundongos , Mutação , Isoformas de Proteínas/genética , Convulsões/genética , Sinapses/fisiologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Animais , Toxina da Cólera , Proteína 4 Homóloga a Disks-Large , Gangliosidose GM1/metabolismo , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas de Membrana/deficiência , Camundongos , Camundongos Transgênicos , Mutação/genética , Domínios PDZ/genética , Domínios PDZ/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Valina/genéticaRESUMO
Glutamate is the major excitatory neurotransmitter in vertebrate and invertebrate nervous systems. Proteins involved in glutamatergic neurotransmission, and chiefly glutamate receptors and their auxiliary subunits, play key roles in nervous system function. Thus, understanding their evolution and uncovering their diversity is essential to comprehend how nervous systems evolved, shaping cognitive function. Comprehensive phylogenetic analysis of these proteins across metazoans have revealed that their evolution is much more complex than what can be anticipated from vertebrate genomes. This is particularly true for ionotropic glutamate receptors (iGluRs), as their current classification into 6 classes (AMPA, Kainate, Delta, NMDA1, NMDA2 and NMDA3) would be largely incomplete. New work proposes a classification of iGluRs into 4 subfamilies that encompass 10 classes. Vertebrate AMPA, Kainate and Delta receptors would belong to one of these subfamilies, named AKDF, the NMDA subunits would constitute another subfamily and non-vertebrate iGluRs would be organised into the previously unreported Epsilon and Lambda subfamilies. Similarly, the animal evolution of metabotropic glutamate receptors has resulted in the formation of four classes of these receptors, instead of the three currently recognised. Here we review our current knowledge on the animal evolution of glutamate receptors and their auxiliary subunits. This article is part of the special issue on 'Glutamate Receptors - Orphan iGluRs'.
Assuntos
Subunidades Proteicas/metabolismo , Receptores de Glutamato/metabolismo , Animais , Evolução Molecular , Ácido Glutâmico/metabolismo , FilogeniaRESUMO
Myoclonus-dystonia (MD) is a rare childhood-onset movement disorder, with an estimated prevalence of about 2 per 1,000,.000 in Europe, characterized by myoclonic jerks in combination with focal or segmental dystonia. Pathogenic variants in the gene encoding ε-sarcoglycan (SGCE), a maternally imprinted gene, are the most frequent genetic cause of MD. To date, the exact role of ε-sarcoglycan and the pathogenic mechanisms that lead to MD are still unknown. However, there are more than 40 reported isoforms of human ε-sarcoglycan, pointing to a complex biology of this protein. Additionally, some of these are brain-specific isoforms, which may suggest an important role within the central nervous system. In the present review, we aim to provide an overview of the current state of knowledge of ε-sarcoglycan. We will focus on the genetic landscape of SGCE and the presence and plausible role of ε-sarcoglycan in the brain. Finally, we discuss the importance of the brain-specific isoforms and hypothesize that SGCE may play essential roles in normal synaptic functioning and their alteration will be strongly related to MD.
Assuntos
Distúrbios Distônicos/genética , Distúrbios Distônicos/metabolismo , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Distúrbios Distônicos/diagnóstico , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinapses/genética , Sinapses/metabolismo , Sinapses/patologiaRESUMO
Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.
Assuntos
Epilepsia/patologia , Hipocampo/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/patologia , Esclerose/genética , Animais , Humanos , CamundongosRESUMO
In 1981 Jeff Watkins and Dick Evans wrote what was to become a seminal review on excitatory amino acids (EAAs) and their receptors (Watkins and Evans, 1981). Bringing together various lines of evidence dating back over several decades on: the distribution in the nervous system of putative amino acid neurotransmitters; enzymes involved in their production and metabolism; the uptake and release of amino acids; binding of EAAs to membranes; the pharmacological action of endogenous excitatory amino acids and their synthetic analogues, and notably the actions of antagonists for the excitations caused by both nerve stimulation and exogenous agonists, often using pharmacological tools developed by Jeff and his colleagues, they provided a compelling account for EAAs, especially l-glutamate, as a bona fide neurotransmitter in the nervous system. The rest, as they say, is history, but far from being consigned to history, EAA research is in rude health well into the 21st Century as this series of Special Issues of Neuropharmacology exemplifies. With EAAs and their receptors flourishing across a wide range of disciplines and clinical conditions, we enter into a dialogue with two of the most prominent and influential figures in the early days of EAA research: Jeff Watkins and Dick Evans.
Assuntos
Aminoácidos Excitatórios/fisiologia , Neurotransmissores/fisiologia , Receptores de Glutamato/fisiologia , Animais , Aminoácidos Excitatórios/farmacologia , Humanos , Receptores de Glutamato/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
In mammalian synapses, the function of ionotropic glutamate receptors is critically modulated by auxiliary subunits. Most of these specifically regulate the synaptic localization and electrophysiological properties of AMPA-type glutamate receptors (AMPARs). Here, we comprehensively investigated the animal evolution of the protein families that contain AMPAR auxiliary subunits (ARASs). We observed that, on average, vertebrates have four times more ARASs than other animal species. We also demonstrated that ARASs belong to four unrelated protein families: CACNG-GSG1, cornichon, shisa and Dispanin C. Our study demonstrates that, despite the ancient origin of these four protein families, the majority of ARASs emerged during vertebrate evolution by independent but convergent processes of neo/subfunctionalization that resulted in the multiple ARASs found in present vertebrate genomes. Importantly, although AMPARs appeared and diversified in the ancestor of bilateral animals, the ARAS expansion did not occur until much later, in early vertebrate evolution. We propose that the surge in ARASs and consequent increase in AMPAR functionalities, contributed to the increased complexity of vertebrate brains and cognitive functions.
Assuntos
Evolução Biológica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Vertebrados , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Genoma , Humanos , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de AMPA/química , Análise de Sequência de DNA , Vertebrados/classificação , Vertebrados/genéticaRESUMO
Autosomal dominant mutations in GRIN2B are associated with severe encephalopathy, but little is known about the pathophysiological outcomes and any potential therapeutic interventions. Genetic studies have described the association between de novo mutations of genes encoding the subunits of the N-methyl-d-aspartate receptor (NMDAR) and severe neurological conditions. Here, we evaluated a missense mutation in GRIN2B, causing a proline-to-threonine switch (P553T) in the GluN2B subunit of NMDAR, which was found in a 5-year-old patient with Rett-like syndrome with severe encephalopathy. Structural molecular modeling predicted a reduced pore size of the mutant GluN2B-containing NMDARs. Electrophysiological recordings in a HEK-293T cell line expressing the mutated subunit confirmed this prediction and showed an associated reduced glutamate affinity. Moreover, GluN2B(P553T)-expressing primary murine hippocampal neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of the AMPA receptor subunit GluA1 at stimulated synapses. Furthermore, the naturally occurring coagonist d-serine restored function to GluN2B(P553T)-containing NMDARs. l-Serine dietary supplementation of the patient was hence initiated, resulting in the increased abundance of d-serine in the plasma and brain. The patient has shown notable improvements in motor and cognitive performance and communication after 11 and 17 months of l-serine dietary supplementation. Our data suggest that l-serine supplementation might ameliorate GRIN2B-related severe encephalopathy and other neurological conditions caused by glutamatergic signaling deficiency.
Assuntos
Encefalopatias , Suplementos Nutricionais , Mutação com Perda de Função , Receptores de N-Metil-D-Aspartato , Síndrome de Rett , Serina , Animais , Encefalopatias/tratamento farmacológico , Encefalopatias/genética , Encefalopatias/metabolismo , Encefalopatias/patologia , Criança , Cognição/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , Serina/administração & dosagem , Serina/farmacocinéticaRESUMO
Synapses are centrally involved in many brain disorders, particularly in psychiatric and neurodevelopmental ones. However, our current understanding of the proteomic alterations affecting synaptic performance in the majority of mental illnesses is limited. As a result, novel pharmacotherapies with improved neurological efficacy have been scarce over the past decades. The main goal of synaptic proteomics in the context of mental illnesses is to identify dysregulated molecular mechanisms underlying these conditions. Here we reviewed and performed a meta-analysis of previous neuroproteomic research to identify proteins that may be consistently dysregulated in one or several mental disorders. Notably, we found very few proteins reproducibly altered among independent experiments for any given condition or between conditions, indicating that we are still far from identifying key pathophysiological mechanisms of mental illness. We suggest that future research in the field will require higher levels of standardization and larger-scale experiments to address the challenge posed by biological and methodological variability. We strongly believe that more resources should be placed in this field as the need to identify the molecular roots of mental illnesses is highly pressing.
Assuntos
Transtornos Mentais , Proteômica , Sinapses/metabolismo , Animais , Humanos , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Transtornos Mentais/patologia , Metanálise como Assunto , Sinapses/patologiaRESUMO
BACKGROUND: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-d-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1+/- mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency. METHODS: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7-8-month old Syngap1+/- mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6days. RESULTS: We found that in hippocampal slices from vehicle-treated Syngap1+/- mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized basal synaptic responses, but did not reverse LTP deficit. CONCLUSIONS: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals.
Assuntos
Benzamidas/farmacologia , Difenilamina/análogos & derivados , Hipocampo/fisiopatologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas Ativadoras de ras GTPase/deficiência , Animais , Difenilamina/farmacologia , Feminino , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Mutação , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Toll-like receptors (TLRs) are important for raising innate immune responses in both invertebrates and vertebrates. Amphioxus belongs to an ancient chordate lineage which shares key features with vertebrates. The genomic research on TLR genes in Branchiostoma floridae and Branchiostoma belcheri reveals the expansion of TLRs in amphioxus. However, the repertoire of TLRs in Branchiostoma lanceolatum has not been studied and the functionality of amphioxus TLRs has not been reported. We have identified from transcriptomic data 30 new putative TLRs in B. lanceolatum and all of them are transcribed in adult amphioxus. Phylogenetic analysis showed that the repertoire of TLRs consists of both non-vertebrate and vertebrate-like TLRs. It also indicated a lineage-specific expansion in orthologous clusters of the vertebrate TLR11 family. We did not detect any representatives of the vertebrate TLR1, TLR3, TLR4, TLR5 and TLR7 families. To gain insight into these TLRs, we studied in depth a particular TLR highly similar to a B. belcheri gene annotated as bbtTLR1. The phylogenetic analysis of this novel BlTLR showed that it clusters with the vertebrate TLR11 family and it might be more related to TLR13 subfamily according to similar domain architecture. Transient and stable expression in HEK293 cells showed that the BlTLR localizes on the plasma membrane, but it did not respond to the most common mammalian TLR ligands. However, when the ectodomain of BlTLR is fused to the TIR domain of human TLR2, the chimeric protein could indeed induce NF-κB transactivation in response to the viral ligand Poly I:C, also indicating that in amphioxus, specific accessory proteins are needed for downstream activation. Based on the phylogenetic, subcellular localization and functional analysis, we propose that the novel BlTLR might be classified as an antiviral receptor sharing at least partly the functions performed by vertebrate TLR22. TLR22 is thought to be viral teleost-specific TLR but here we demonstrate that teleosts and amphioxus TLR22-like probably shared a common ancestor. Additional functional studies with other lancelet TLR genes will enrich our understanding of the immune response in amphioxus and will provide a unique perspective on the evolution of the immune system.
Assuntos
Anfioxos/genética , RNA de Cadeia Dupla/genética , Receptores Toll-Like/genética , Animais , Linhagem Celular , Membrana Celular/genética , Genoma/genética , Genômica/métodos , Células HEK293 , Humanos , Imunidade Inata/genética , Mamíferos/genética , NF-kappa B/genética , Filogenia , Análise de Sequência de DNA/métodos , Especificidade da Espécie , Ativação Transcricional/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR. METHODS: We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice. RESULTS: Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17 months of D-serine dietary supplementation. CONCLUSIONS: Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.
RESUMO
Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings.