RESUMO
Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Plasticidade Neuronal , Densidade Pós-Sináptica/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Proteína 4 Homóloga a Disks-Large , Células HEK293 , Células HeLa , Hipocampo/citologia , Hipocampo/embriologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Camundongos , Neurônios/metabolismo , Transição de Fase , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Ratos , Proteínas Ativadoras de ras GTPase/químicaRESUMO
SYNGAP1 is a Ras-GTPase-activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDDs). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), an NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances. Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning, and memory and have seizures. However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A. While reduction in Syngap1 mRNA varies from 30 to 50% depending on the specific mutation, both models show ~50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder.
Assuntos
Epilepsia , Deficiência Intelectual , Humanos , Animais , Camundongos , Códon sem Sentido , Convulsões , Encéfalo , Modelos Animais de Doenças , Transtornos da Memória , Proteínas Ativadoras de ras GTPaseRESUMO
Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene-mutation-cell-behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and â¼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and â¼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine.
Assuntos
Sistema de Sinalização das MAP Quinases/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Transmissão Sináptica/genética , Animais , Células Cultivadas , Doença/genética , Feminino , Técnicas de Transferência de Genes , Humanos , Lentivirus/genética , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Ratos Sprague-Dawley , Técnicas de Cultura de TecidosRESUMO
AIM: The anti-programmed death-ligand 1 antibody atezolizumab and vascular endothelial growth factor-neutralizing antibody bevacizumab in combination (Atezo + Bev) have become the first-line therapy in advanced hepatocellular carcinoma (HCC). Distinct types of tumor immune microenvironment (TIME) and their associations with specific molecular subclasses and driver gene mutations have been identified in HCC; however, these insights are mainly based on surgically resected early-stage tumors. The current study aimed to reveal the biology and TIME of advanced HCC and their significance in predicting clinical outcomes of Atezo + Bev therapy. METHODS: Thirty-three patients with advanced HCC who were scheduled for treatment with Atezo + Bev therapy were included in this study. Pretreatment tumor biopsy, pre- and posttreatment diffusion-weighted magnetic resonance imaging (MRI) with nine b values (0-1500 s/mm2 ), and other clinicopathologic factors were analyzed. RESULTS: Compared with resectable HCC, advanced HCC was characterized by higher proliferative activity, a higher frequency of Wnt/ß-catenin-activated HCC, and lower lymphocytic infiltration. Prognostically, two metabolism-related factors, histopathologically determined tumor steatosis and/or glutamine synthetase (GS) expression, and MRI-determined tumor steatosis, were the most significant prognostic indicators for progression-free survival (PFS) and overall survival after Atezo + Bev therapy. Furthermore, changes in the pre- and posttreatment true diffusion coefficients on MRI, which might reflect changes in TIME after treatment, were significantly associated with better PFS. CONCLUSIONS: The biology and TIME of HCC were strikingly different in advanced HCC compared with those of surgically resected HCC. Two metabolism-related factors, pathologically determined tumor steatosis and/or GS expression, and MRI-determined tumor steatosis, were found to be the most significant prognostic indicators for Atezo + Bev therapy in advanced HCC.
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SynGAP is a potent regulator of biochemical signaling in neurons and plays critical roles in neuronal function. It was first identified in 1998, and has since been extensively characterized as a mediator of synaptic plasticity. Because of its involvement in synaptic plasticity, SynGAP has emerged as a critical protein for normal cognitive function. In recent years, mutations in the SYNGAP1 gene have been shown to cause intellectual disability in humans and have been linked to other neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia. While the structure and biochemical function of SynGAP have been well characterized, a unified understanding of the various roles of SynGAP at the synapse and its contributions to neuronal function remains to be achieved. In this review, we summarize and discuss the current understanding of the multifactorial role of SynGAP in regulating neuronal function gathered over the last two decades.
Assuntos
Encéfalo/fisiologia , Cognição/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Proteínas Ativadoras de ras GTPase/fisiologia , Animais , Humanos , Plasticidade Neuronal/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
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
Background Recently, diffusion-weighted imaging (DWI) and quantitative enhancement ratio measured at the hepatobiliary phase (HBP) of Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) has been established as an effective method for evaluating liver fibrosis. Purpose To evaluate which is a more favorable surrogate marker in predicting high-stage liver fibrosis, apparently diffusion coefficient (ADC) value or quantitative enhancement ratio measured on HBP. Material and Methods Eighty-three patients with 99 surgically resected hepatic lesions were enrolled in this study. DWI was performed with b-values of 100 and 800 s/mm2. Regions of interest were set on ADC map, and the HBP of Gd-EOB-DTPA-enhanced MRI, to calculate ADC value, liver-to-muscle ratio (LMR), liver-to-spleen ratio (LSR), and contrast enhancement index (CEI) of liver. We compared these parameters between low-stage fibrosis (F0, F1, and F2) and high-stage fibrosis (F3 and F4). Receiver operating characteristic analysis was performed to compare the diagnostic performance when distinguishing low-stage fibrosis from high-stage fibrosis. Results LMR and CEI were significantly lower at high-stage fibrosis than at the low stage ( P < 0.01 and P = 0.04, respectively), whereas LSR did not show a significant difference ( P = 0.053). No significant difference was observed in diagnostic performance between LMR and CEI ( P = 0.185). The best sensitivity and specificity, when an LMR of 2.80 or higher was considered to be low-stage fibrosis, were 82.4% and 75.6%, respectively. ADC value showed no significant differences among fibrosis grades ( P = 0.320). Conclusion LMR and CEI were both adequate surrogate parameters to distinguish high-stage fibrosis from low-stage fibrosis.
Assuntos
Meios de Contraste/administração & dosagem , Imagem de Difusão por Ressonância Magnética/métodos , Gadolínio DTPA/administração & dosagem , Aumento da Imagem/métodos , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/patologia , Neoplasias Hepáticas/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. Reduction in the dendritic spine density is a major alteration that has been reproducibly reported in the cerebral cortex of patients with schizophrenia. Disrupted-in-Schizophrenia-1 (DISC1), a factor that influences endophenotypes underlying schizophrenia and several other neuropsychiatric disorders, has a regulatory role in the postsynaptic density in association with the NMDA-type glutamate receptor, Kalirin-7, and Rac1. Prolonged knockdown of DISC1 leads to synaptic deterioration, reminiscent of the synaptic pathology of schizophrenia. Thus, we tested the effects of novel inhibitors to p21-activated kinases (PAKs), major targets of Rac1, on synaptic deterioration elicited by knockdown expression of DISC1. These compounds not only significantly ameliorated the synaptic deterioration triggered by DISC1 knockdown but also partially reversed the size of deteriorated synapses in culture. One of these PAK inhibitors prevented progressive synaptic deterioration in adolescence as shown by in vivo two-photon imaging and ameliorated a behavioral deficit in prepulse inhibition in adulthood in a DISC1 knockdown mouse model. The efficacy of PAK inhibitors may have implications in drug discovery for schizophrenia and related neuropsychiatric disorders in general.
Assuntos
Envelhecimento/patologia , Espinhas Dendríticas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Esquizofrenia/tratamento farmacológico , Esquizofrenia/enzimologia , Quinases Ativadas por p21/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/enzimologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Piridonas/química , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Interferência de RNA/efeitos dos fármacos , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/fisiopatologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid ß-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aß generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aß in vivo.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas de Ligação ao Cálcio/genética , Secretases da Proteína Precursora do Amiloide/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Caderinas , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso , Estrutura Terciária de Proteína , Proteólise , Via Secretória/genéticaRESUMO
Kinesin-1 anterogradely transports vesicles containing cargo proteins when a protein-protein interaction activates it from an inhibited state. The C-terminal cytoplasmic region of kinesin-1 cargo protein Alcadeinα (Alcα) interacts with the KLC1 subunit's tetratricopeptide repeat (TPR) region, activating kinesin-1's association with vesicles and anterograde transport. We found that either of two 10-amino-acid WD motifs in Alcα cytoplasmic region was necessary and sufficient to initiate this activation. An artificial transmembrane protein containing either WD motif induced kinesin-1's vesicular association and anterograde transport in a KLC-dependent manner, even in the normally inhibiting presence of excess KLC1, thus allowing us to analyze the KLC1 TPR-WD functional interaction in detail in vivo. A part of TPR region was dispensable for the WD motifs' activation of kinesin-1 and transport, indicating that only part of the TPR structure is required for this function in vivo. For a different kinesin-1 cargo protein, JIP1, an 11-amino-acid C-terminal region was sufficient to recruit KLC1 to vesicles, but did not activate transport. These observations suggest that structurally different TPR-interacting peptides may have different effects on kinesin-1. This mechanism may partly explain how kinesin-1 can organize the transport of a wide variety of cargo molecules.
Assuntos
Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/química , Peptídeos/química , Motivos de Aminoácidos , Animais , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinesinas/química , Camundongos , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Frações Subcelulares/metabolismoRESUMO
Homeostatic responses critically adjust synaptic strengths to maintain stability in neuronal networks. Compensatory adaptations to prolonged excitation include induction of Polo-like kinases (Plks) and degradation of spine-associated Rap GTPase-activating protein (SPAR) to reduce synaptic excitation, but mechanisms that limit overshooting and allow refinement of homeostatic adjustments remain poorly understood. We report that Plks produce canonical pathway-mediated activation of the nuclear factor κB (NF-κB) transcription factor in a process that requires the kinase activity of Plks. Chronic elevated activity, which induces Plk expression, also produces Plk-dependent activation of NF-κB. Deficiency of NF-κB, in the context of exogenous Plk2 expression or chronic elevated neuronal excitation, produces exaggerated homeostatic reductions in the size and density of dendritic spines, synaptic AMPA glutamate receptor levels, and excitatory synaptic currents. During the homeostatic response to chronic elevated activity, NF-κB activation by Plks subsequently opposes Plk-mediated SPAR degradation by transcriptionally upregulating SPAR in mouse hippocampal neurons in vitro and in vivo. Exogenous SPAR expression can rescue the overshooting of homeostatic reductions at excitatory synapses in NF-κB-deficient neurons responding to elevated activity. Our data establish an integral feedback loop involving NF-κB, Plks, and SPAR that regulates the end point of homeostatic synaptic adaptation to elevated activity and are the first to implicate a transcription factor in the regulation of homeostatic synaptic responses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Homeostase/fisiologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Hipocampo/metabolismo , Camundongos , Neurônios/metabolismo , Fosforilação , Receptores de AMPA/metabolismo , Quinase 1 Polo-LikeRESUMO
Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.
Assuntos
Ácido Glutâmico/genética , Deficiência Intelectual/genética , Mutação/genética , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Células HEK293 , Humanos , Cinesinas/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fenótipo , Ligação Proteica/genética , Transporte Proteico , Splicing de RNA/genética , Ratos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Frações Subcelulares/metabolismo , SíndromeRESUMO
OBJECTIVE: To investigate if tracer kinetic modelling of low temporal resolution dynamic contrast-enhanced (DCE) MRI with Gd-EOB-DTPA could replace technetium-99 m galactosyl human serum albumin (GSA) single positron emission computed tomography (SPECT) and indocyanine green (ICG) retention for the measurement of liver functional reserve. METHODS: Twenty eight patients awaiting liver resection for various cancers were included in this retrospective study that was approved by the institutional review board. The Gd-EOB-DTPA MRI sequence acquired five images: unenhanced, double arterial phase, portal phase, and 4 min after injection. Intracellular contrast uptake rate (UR) and extracellular volume (Ve) were calculated from DCE-MRI, along with the ratio of GSA radioactivity of liver to heart-plus-liver and per cent of cumulative uptake from 15-16 min (LHL15 and LU15, respectively) from GSA-scintigraphy. ICG retention at 15 min, Child-Pugh cirrhosis score (CPS) and postoperative Inuyama fibrosis criteria were also recorded. Statistical analysis was with Spearman rank correlation analysis. RESULTS: Comparing MRI parameters with the reference methods, significant correlations were obtained for UR and LHL15, LU15, ICG15 (all 0.4-0.6, P < 0.05); UR and CPS (-0.64, P < 0.001); Ve and Inuyama (0.44, P < 0.05). CONCLUSION: Measures of liver function obtained by routine Gd-EOB-DTPA DCE-MRI with tracer kinetic modelling may provide a suitable method for the evaluation of liver functional reserve. KEY POINTS: ⢠Magnetic resonance imaging (MRI) provides new methods of measuring hepatic functional reserve. ⢠DCE-MRI with Gd-EOB-DTPA offers the possibility of replacing scintigraphy. ⢠The analysis method can be used for preoperative liver function evaluation.
Assuntos
Gadolínio DTPA , Verde de Indocianina , Neoplasias Hepáticas/diagnóstico , Imageamento por Ressonância Magnética/métodos , Agregado de Albumina Marcado com Tecnécio Tc 99m , Pentetato de Tecnécio Tc 99m , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Idoso , Corantes , Meios de Contraste , Feminino , Seguimentos , Hepatectomia , Humanos , Fígado/diagnóstico por imagem , Fígado/patologia , Testes de Função Hepática , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/cirurgia , Masculino , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Background and aims: SYNGAP1-related disorder (SYNGAP1-RD) is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. Methods: We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild-type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. Results: We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab mutant larvae are hyperactive compared to wild-type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, and hyperactivity was proportional to the number of mutant syngap1 alleles. Limitations: Syngap1 loss-of-function mutations produce relatively subtle phenotypes in zebrafish compared to mammals. For example, while mouse Syngap1 homozygotes die at birth, zebrafish syngap1ab-/- survive to adulthood and are fertile, thus some aspects of symptoms in people with SYNGAP1-Related Disorder are not likely to be reflected in zebrafish. Conclusion: Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.
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To investigate changes in skeletal muscle mass and fat fraction in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes mellitus (T2DM) undergoing treatment with Semaglutide for 6months. This single-arm pilot study included 21 patients with MASLD who received semaglutide for T2DM. Body weight, metabolic parameters, liver enzymes, fibrosis markers, skeletal muscle index (cm2/m2), and fat fraction (%) at the L3 level using the two-point Dixon method on magnetic resonance imaging (MRI), as well as liver steatosis and liver stiffness assessed using MRI-based proton density fat fraction (MRI-PDFF) and MR elastography, respectively, were prospectively examined before and 6 months after semaglutide administration. The mean age of the patients was 53 years and 47.6% were females. The median liver steatosis-fraction (%) and skeletal muscle steatosis-fraction values (%) significantly decreased (22.0 vs 12.0; Pâ =â .0014) and (12.8 vs 9.9; Pâ =â .0416) at baseline and 6 months, respectively, while maintaining muscle mass during treatment. Semaglutide also dramatically reduced hemoglobin A1c (%) (6.8 vs 5.8, Pâ =â .0003), AST (IU/L) (54 vs 26, Pâ <â .0001), ALT (IU/L) (80 vs 34, Pâ =â .0004), and γ-GTP (IU/L) levels (64 vs 34, Pâ =â .0007). Although not statistically significant, Body weight (kg) (79.9 vs 77.4), body mass index (BMI) (kg/m2) (28.9 vs 27.6), and liver stiffness (kPa) (28.9 vs 27.6) showed a decreasing trend. Fibrosis markers such as M2BPGi, type IV collagen, and skeletal muscle area did not differ. Semaglutide demonstrated favorable effects on liver and skeletal muscle steatosis, promoting improved liver function and diabetic status.
Assuntos
Diabetes Mellitus Tipo 2 , Peptídeos Semelhantes ao Glucagon , Fígado , Músculo Esquelético , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Diabetes Mellitus Tipo 2/tratamento farmacológico , Estudos Prospectivos , Músculo Esquelético/efeitos dos fármacos , Peptídeos Semelhantes ao Glucagon/uso terapêutico , Peptídeos Semelhantes ao Glucagon/administração & dosagem , Projetos Piloto , Fígado/efeitos dos fármacos , Fígado/diagnóstico por imagem , Fígado/patologia , Hipoglicemiantes/uso terapêutico , Fígado Gorduroso/tratamento farmacológico , Adulto , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Imageamento por Ressonância Magnética , Técnicas de Imagem por Elasticidade , Hemoglobinas Glicadas/efeitos dos fármacos , Hemoglobinas Glicadas/análise , IdosoRESUMO
SynGAP is an abundant synaptic GTPase-activating protein (GAP) critical for synaptic plasticity, learning, memory, and cognition. Mutations in SYNGAP1 in humans result in intellectual disability, autistic-like behaviors, and epilepsy. Heterozygous Syngap1-knockout mice display deficits in synaptic plasticity, learning, and memory and exhibit seizures. It is unclear whether SynGAP imparts structural properties at synapses independently of its GAP activity. Here, we report that inactivating mutations within the GAP domain do not inhibit synaptic plasticity or cause behavioral deficits. Instead, SynGAP modulates synaptic strength by physically competing with the AMPA-receptor-TARP excitatory receptor complex in the formation of molecular condensates with synaptic scaffolding proteins. These results have major implications for developing therapeutic treatments for SYNGAP1-related neurodevelopmental disorders.
Assuntos
Cognição , Plasticidade Neuronal , Proteínas Ativadoras de ras GTPase , Animais , Humanos , Camundongos , Transtorno Autístico/genética , Proteínas Ativadoras de GTPase/genética , Aprendizagem , Camundongos Knockout , Plasticidade Neuronal/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , CatáliseRESUMO
PURPOSE: To evaluate liver function obtained by tracer-kinetic modeling of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data acquired with a routine gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced protocol. MATERIALS AND METHODS: Data were acquired from 25 cases of nonchronic liver disease and 94 cases of cirrhosis. DCE-MRI was performed with a dose of 0.025 mmol/kg Gd-EOB-DTPA injected at 2 mL/sec. A 3D breath-hold sequence acquired 5 volumes of 72 slices each: precontrast, double arterial phase, portal phase, and 4-minute postcontrast. Regions of interest (ROIs) were selected semiautomatically in the aorta, portal vein, and whole liver on a middle slice. A constrained dual-inlet two-compartment uptake model was fitted to the ROI curves, producing three parameters: intracellular uptake rate (UR), extracellular volume (Ve), and arterial flow fraction (AFF). RESULTS: Median UR dropped from 4.46 10(-2) min(-1) in the noncirrhosis to 3.20 in Child-Pugh A (P = 0.001), and again to 1.92 in Child-Pugh B (P < 0.0001). Median Ve dropped from 6.64 mL 100 mL(-1) in the noncirrhosis to 5.80 in Child-Pugh A (P = 0.01). Other combinations of Ve and AFF changes were not significant for any group. CONCLUSION: UR obtained from tracer kinetic analysis of a routine DCE-MRI has the potential to become a novel index of liver function.
Assuntos
Doença Hepática Terminal/diagnóstico , Doença Hepática Terminal/metabolismo , Gadolínio DTPA/farmacocinética , Interpretação de Imagem Assistida por Computador/métodos , Testes de Função Hepática/métodos , Imageamento por Ressonância Magnética/métodos , Idoso , Algoritmos , Simulação por Computador , Meios de Contraste/farmacocinética , Feminino , Humanos , Aumento da Imagem/métodos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.
Assuntos
Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Receptores de AMPA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Membrana Celular/metabolismo , Células Cultivadas , Primers do DNA/genética , Hipocampo/citologia , Hipocampo/metabolismo , Dados de Sequência Molecular , Plasticidade Neuronal , Neurônios/metabolismo , Multimerização Proteica , Subunidades Proteicas , Edição de RNA , Ratos , Receptores de AMPA/química , Receptores de AMPA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Background and Aims: SYNGAP1 disorder is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. Methods: We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. Results: We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab larvae are hyperactive compared to wild type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, with overall movement increasing with the number of mutant syngap1 alleles. Conclusions: Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.