RESUMO
AIMS: Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance. METHODS: Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib. RESULTS: SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1ß inhibition. CONCLUSIONS: SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Sirtuínas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Inflamassomos/metabolismo , Inflamassomos/farmacologia , Fosforilação , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sistema de Sinalização das MAP Quinases , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/farmacologia , Sirtuínas/genética , Sirtuínas/metabolismo , Sirtuínas/farmacologiaRESUMO
Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.
Assuntos
Ferroptose , Neoplasias , Humanos , 1-Alquil-2-acetilglicerofosfocolina Esterase/antagonistas & inibidores , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias/tratamento farmacológicoRESUMO
ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.
Assuntos
Transtorno Autístico , Deficiência Intelectual , Camundongos , Animais , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/genética , Potenciação de Longa Duração/genética , Transtorno Autístico/metabolismo , Cognição , Proteínas de Homeodomínio/metabolismoRESUMO
PURPOSE: Albumin-bilirubin (ALBI) score is a well-known prognostic factor for various diseases, including colorectal cancer (CRC). However, little is known about the significance of postoperative ALBI score changes in patients with CRC. MATERIALS AND METHODS: A total of 723 patients who underwent surgery were enrolled. Preoperative ALBI (ALBI-pre) and postoperative ALBI (ALBI-post) scores were divided into low and high score groups. ALBI-trend was defined as a combination of four groups comprising the low and high ALBI-pre and ALBI-post score groups. Kaplan-Meier survival curves were used to compare the overall survival (OS) between the different ALBI groups. The Cox proportional hazards model was used to examine the independent relevant factors of OS. Stratification performance was compared between the different ALBI groupings using Harrell's concordance index (C-index). RESULTS: ALBI-pre, ALBI-post, and ALBI-trend score groups were significant prognostic factors of OS in the univariable analysis. However, multivariable analysis showed that ALBI-trend was an independent prognostic factor while ALBI-pre and ALBI-post were not. The C-index of ALBI-trend (0.622; 95% confidence interval [CI], 0.587 to 0.655) was higher than that of ALBI-pre (0.589; 95% CI, 0.557 to 0.621; bootstrap mean difference, 0.033; 95% CI, 0.013 to 0.057) and ALBI-post (0.575; 95% CI, 0.545 to 0.605; bootstrap mean difference, 0.047; 95% CI, 0.024 to 0.074). CONCLUSION: Combining ALBI-pre and ALBI-post scores is an independent prognostic factor of OS and shows superior predictive power compared to ALBI-pre or ALBI-post alone in patients with CRC.
Assuntos
Bilirrubina , Neoplasias Colorretais , Albumina Sérica , Humanos , Relevância Clínica , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Prognóstico , Estudos RetrospectivosRESUMO
The molecular basis of Z-DNA recognition and stabilization is mostly discovered via X-ray crystallography. The sequences composed with alteration of purine and pyrimidine are known to adopt Z-DNA conformation. Due to the energy penalty for forming Z-DNA, the small molecular stabilizer or Z-DNA-specific binding protein is required for DNA to adopt Z conformation prior to crystallizing Z-DNA. Here we described the methods ranging from preparation of DNA and Z-alpha protein to crystallization of Z-DNA in detail.
Assuntos
DNA Forma Z , Cristalização , Modelos Moleculares , Sequência de Bases , DNA/química , Conformação de Ácido Nucleico , Cristalografia por Raios XRESUMO
The left-handed Z-DNA is surrounded by right-handed canonical B-DNA, and thus the junction between B- and Z-DNA has been occurred during temporal Z-DNA formation in the genome. The base extrusion structure of the BZ junction may help detect Z-DNA formation in DNAs. Here we describe the BZ junction structural detection by using 2-aminopurine (2AP) fluorescent probe. BZ junction formation can be measured in solution by this method.
Assuntos
DNA de Forma B , DNA Forma Z , DNA/genética , 2-Aminopurina/química , Replicação do DNA , Conformação de Ácido NucleicoRESUMO
Parkinson's disease (PD) belongs to an α-synucleinopathy and manifests motor dysfunction attributed to nigrostriatal dopaminergic degeneration. In clinical practice, the beneficial role of physical therapy such as motor skill learning training has been recognized in PD-linked motor defects. Nevertheless, the disease-modifying effects of motor skill learning training on PD-related pathology remain unclear. Here, we investigated the disease-modifying effects of rotarod walking exercise (RWE), a modality of motor skill learning training, in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In motor function and dopaminergic degeneration, RWE improved MPTP-induced deficits. In addition, RWE enhanced the expression of neurotrophic factors BDNF/GDNF, PGC1-α, Nurr1, and p-AMPK, thereby recovering dopaminergic neuronal cell death. Moreover, RWE inhibited microglial activation and the expression of pro-inflammatory markers, such as p-IκBα, iNOS, IL-1ß, TNF-α, and cathepsin D, while elevating anti-inflammatory IL-10 and TGF-ß. RWE also decreased oxidative stress markers in the substantia nigra, such as 4-HNE and 8-OHdG-positive cells, while increasing Nrf2-controlled antioxidant enzymes. Regarding the effect of RWE on α-synuclein, it reduced the monomer/oligomer forms of α-synuclein and phosphorylation at serine 129. Further mechanistic studies revealed that RWE suppressed the expression of matrix metalloproteinase-3 and p-GSK3ß (Y216), which play key roles in α-synuclein aggregation. These data collectively suggest that inhibition of neuroinflammation and α-synuclein oligomerization by RWE may contribute to the improvement of PD pathology.
Assuntos
Doença de Parkinson , Animais , Camundongos , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Doenças Neuroinflamatórias , Substância Negra , Dopamina/metabolismo , Caminhada , Camundongos Endogâmicos C57BL , Neurônios Dopaminérgicos , Modelos Animais de Doenças , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologiaRESUMO
Autism spectrum disorder is characterized by early postnatal symptoms, although little is known about the mechanistic deviations that produce them and whether correcting them has long-lasting preventive effects on adult-stage deficits. ARID1B, a chromatin remodeler implicated in neurodevelopmental disorders, including autism spectrum disorder, exhibits strong embryonic- and early postnatal-stage expression. We report here that Arid1b-happloinsufficient (Arid1b+/-) mice display autistic-like behaviors at juvenile and adult stages accompanied by persistent decreases in excitatory synaptic density and transmission. Chronic treatment of Arid1b+/- mice with fluoxetine, a selective serotonin-reuptake inhibitor, during the first three postnatal weeks prevents synaptic and behavioral deficits in adults. Mechanistically, these rescues accompany transcriptomic changes, including upregulation of FMRP targets and normalization of HDAC4/MEF2A-related transcriptional regulation of the synaptic proteins, SynGAP1 and Arc. These results suggest that chronic modulation of serotonergic receptors during critical early postnatal periods prevents synaptic and behavioral deficits in adult Arid1b+/- mice through transcriptional reprogramming.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Serotonina , Fatores de Transcrição , Animais , Fluoxetina , Haploinsuficiência , Camundongos , Serotonina/metabolismo , Fatores de Transcrição/genética , Proteínas Ativadoras de ras GTPaseRESUMO
The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Astrócitos , Adesões Focais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Astrócitos/metabolismo , Proteínas de Transporte/genética , Adesões Focais/metabolismo , CamundongosRESUMO
Protein lysine acetylation is a post-translational modification that regulates protein structure and function. It is targeted to proteins by lysine acetyltransferases (KATs) or removed by lysine deacetylases. This work identifies a role for the KAT enzyme general control of amino acid synthesis protein 5 (GCN5; KAT2A) in regulating muscle integrity by inhibiting DNA binding of the transcription factor/repressor Yin Yang 1 (YY1). Here we report that a muscle-specific mouse knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. GCN5 was found to acetylate YY1 at two residues (K392 and K393), disrupting the interaction between the YY1 zinc finger region and DNA. These findings were supported by human data, including an observed negative correlation between YY1 gene expression and muscle fiber diameter. Collectively, GCN5 positively regulates muscle integrity through maintenance of structural protein expression via acetylation-dependent inhibition of YY1. This work implicates the role of protein acetylation in the regulation of muscle health and for consideration in the design of novel therapeutic strategies to support healthy muscle during myopathy or aging.
Assuntos
Distrofina/genética , Músculos/metabolismo , Fator de Transcrição YY1/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Envelhecimento/metabolismo , Animais , DNA/metabolismo , Distrofina/metabolismo , Regulação da Expressão Gênica , Humanos , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Músculos/patologia , Músculos/ultraestrutura , Atrofia Muscular/patologia , Distrofias Musculares/patologia , Transcriptoma/genética , Fatores de Transcrição de p300-CBP/deficiênciaRESUMO
Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.
Assuntos
Glicina , Receptores de N-Metil-D-Aspartato , Animais , Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Homeostase , Proteínas de Membrana Transportadoras , Camundongos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The novel coronavirus, SARS-CoV-2, or 2019-nCoV, which originated in Wuhan, Hubei province, China in December 2019, is a grave threat to public health worldwide. A total of 3,672,238 confirmed cases of coronavirus disease 2019 (COVID-19) and 254,045 deaths were reported globally up to May 7, 2020. However, approved antiviral agents for the treatment of patients with COVID-19 remain unavailable. Drug repurposing of approved antivirals against other viruses such as HIV or Ebola virus is one of the most practical strategies to develop effective antiviral agents against SARS-CoV-2. A combination of repurposed drugs can improve the efficacy of treatment, and structure-based drug design can be employed to specifically target SARS-CoV-2. This review discusses therapeutic strategies using promising antiviral agents against SARS-CoV-2. In addition, structural characterization of potentially therapeutic viral or host cellular targets associated with COVID-19 have been discussed to refine structure-based drug design strategies.
RESUMO
Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is essential for human development, and DYRK1A haploinsufficiency is associated with a recognizable developmental syndrome and variable clinical features. Here, we present a patient with DYRK1A haploinsufficiency syndrome, including facial dysmorphism, delayed motor development, cardiovascular system defects, and brain atrophy. Exome sequencing identified a novel de novo heterozygous mutation of the human DYRK1A gene (c.1185dup), which generated a translational termination codon and resulted in a C-terminally truncated protein (DYRK1A-E396ter). To study the molecular effect of this truncation, we generated mammalian cell and Drosophila models that recapitulated the DYRK1A protein truncation. Analysis of the structure and deformation energy of the mutant protein predicted a reduction in protein stability. Experimentally, the mutant protein was efficiently degraded by the ubiquitin-dependent proteasome pathway and was barely detectable in mammalian cells. More importantly, the mutant kinase was intrinsically inactive and had little negative impact on the wild-type protein. Similarly, the mutant protein had a minimal effect on Drosophila phenotypes, confirming its loss-of-function in vivo. Together, our results suggest that the novel heterozygous mutation of DYRK1A resulted in loss-of-function of the kinase activity of DYRK1A and may contribute to the developmental delay observed in the patient.
Assuntos
Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/genética , Heterozigoto , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Drosophila melanogaster , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Simulação de Dinâmica Molecular , Linhagem , Conformação Proteica em Folha beta , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Quinases DyrkRESUMO
Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.
Assuntos
Ansiedade/genética , Hipocampo/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Animais , Ansiedade/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Ciclosserina/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas de Introdução de Genes , Haploinsuficiência/genética , Heterozigoto , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Synaptic adhesion molecules regulate synapse development and function. However, whether and how presynaptic adhesion molecules regulate postsynaptic NMDAR function remains largely unclear. Presynaptic LAR family receptor tyrosine phosphatases (LAR-RPTPs) regulate synapse development through mechanisms that include trans-synaptic adhesion; however, whether they regulate postsynaptic receptor functions remains unknown. Here we report that presynaptic PTPσ, a LAR-RPTP, enhances postsynaptic NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity in the hippocampus. This regulation does not involve trans-synaptic adhesions of PTPσ, suggesting that the cytoplasmic domains of PTPσ, known to have tyrosine phosphatase activity and mediate protein-protein interactions, are important. In line with this, phosphotyrosine levels of presynaptic proteins, including neurexin-1, are strongly increased in PTPσ-mutant mice. Behaviorally, PTPσ-dependent NMDAR regulation is important for social and reward-related novelty recognition. These results suggest that presynaptic PTPσ regulates postsynaptic NMDAR function through trans-synaptic and direct adhesion-independent mechanisms and novelty recognition in social and reward contexts.
Assuntos
Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/fisiologia , Animais , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Transgênicos , Neuroimagem , Teste de Campo Aberto , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Receptores de N-Metil-D-Aspartato/genética , Transmissão Sináptica/fisiologiaRESUMO
Synaptic adhesion molecules regulate synapse development through trans-synaptic adhesion and assembly of diverse synaptic proteins. Many synaptic adhesion molecules positively regulate synapse development; some, however, exert negative regulation, although such cases are relatively rare. In addition, synaptic adhesion molecules regulate the amplitude of post-synaptic receptor responses, but whether adhesion molecules can regulate the kinetic properties of post-synaptic receptors remains unclear. Here we report that Clmp, a homophilic adhesion molecule of the Ig domain superfamily that is abundantly expressed in the brain, reaches peak expression at a neonatal stage (week 1) and associates with subunits of AMPA receptors (AMPARs) and kainate receptors (KARs). Clmp deletion in mice increased the frequency and amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) and the frequency, amplitude, and decay time constant of KAR-mediated mEPSCs in hippocampal CA3 neurons. Clmp deletion had minimal impacts on evoked excitatory synaptic currents at mossy fiber-CA3 synapses but increased extrasynaptic KAR, but not AMPAR, currents, suggesting that Clmp distinctly inhibits AMPAR and KAR responses. Behaviorally, Clmp deletion enhanced novel object recognition and susceptibility to kainate-induced seizures, without affecting contextual or auditory cued fear conditioning or pattern completion-based contextual fear conditioning. These results suggest that Clmp negatively regulates hippocampal excitatory synapse development and AMPAR and KAR responses in the neonatal hippocampal CA3 as well as object recognition and kainate seizure susceptibility in mice.
RESUMO
Mutations in Tbr1, a high-confidence ASD (autism spectrum disorder)-risk gene encoding the transcriptional regulator TBR1, have been shown to induce diverse ASD-related molecular, synaptic, neuronal, and behavioral dysfunctions in mice. However, whether Tbr1 mutations derived from autistic individuals cause similar dysfunctions in mice remains unclear. Here we generated and characterized mice carrying the TBR1-K228E de novo mutation identified in human ASD and identified various ASD-related phenotypes. In heterozygous mice carrying this mutation (Tbr1 +/K228E mice), levels of the TBR1-K228E protein, which is unable to bind target DNA, were strongly increased. RNA-Seq analysis of the Tbr1 +/K228E embryonic brain indicated significant changes in the expression of genes associated with neurons, astrocytes, ribosomes, neuronal synapses, and ASD risk. The Tbr1 +/K228E neocortex also displayed an abnormal distribution of parvalbumin-positive interneurons, with a lower density in superficial layers but a higher density in deep layers. These changes were associated with an increase in inhibitory synaptic transmission in layer 6 pyramidal neurons that was resistant to compensation by network activity. Behaviorally, Tbr1 +/K228E mice showed decreased social interaction, increased self-grooming, and modestly increased anxiety-like behaviors. These results suggest that the human heterozygous TBR1-K228E mutation induces ASD-related transcriptomic, protein, neuronal, synaptic, and behavioral dysfunctions in mice.