RESUMO
Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.
Assuntos
Transportadores de Ânions Orgânicos , Ácido Úrico , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Ácido Ascórbico/metabolismo , Transporte Biológico , Mamíferos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/genética , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Ácido Úrico/metabolismoRESUMO
Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.
Assuntos
Encéfalo , Callithrix , Animais , Encéfalo/fisiologia , Neurônios , Neurogênese , Células-Tronco EmbrionáriasRESUMO
Recent genome-wide association studies have revealed some genetic loci associated with serum uric acid levels and susceptibility to gout/hyperuricemia which contain potential candidates of physiologically important urate transporters. One of these novel loci is located upstream of SGK1 and SLC2A12, suggesting that variations in these genes increase the risks of hyperuricemia and gout. We herein focused on SLC2A12 encoding a transporter, GLUT12, the physiological function of which remains unclear. As GLUT12 belongs to the same protein family as a well-recognized urate transporter GLUT9, we hypothesized that GLUT12 mediates membrane transport of urate. Therefore, we conducted functional assays and analyzed Glut12 knockout hyperuricemia model mice, generated using the CRISPR-Cas9 system. Our results revealed that GLUT12 acts as a physiological urate transporter and its dysfunction elevates the blood urate concentration. This study provides insights into the deeper understanding of the urate regulatory system in the body, which is also important for pathophysiology of gout/hyperuricemia.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hiperuricemia/sangue , Ácido Úrico/sangue , Animais , Regulação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/genética , Camundongos , Camundongos Knockout , Ácido Úrico/metabolismoRESUMO
The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.
Assuntos
Sinalização do Cálcio , Núcleo Celular/metabolismo , Criopreservação , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Espermátides/metabolismo , Animais , Callithrix , Feminino , Masculino , Camundongos , Microinjeções , Oócitos/citologia , Espermátides/citologiaRESUMO
Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.
Assuntos
Células Ciliadas Auditivas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Técnicas Biossensoriais , Cóclea/citologia , Cóclea/metabolismo , Cães , Transferência Ressonante de Energia de Fluorescência , Humanos , Imuno-Histoquímica , Hibridização In Situ , Células Madin Darby de Rim Canino , Camundongos , Microscopia Eletroquímica de Varredura , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos/métodos , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages.
Assuntos
Tamanho Corporal/fisiologia , Cartilagem/citologia , Cartilagem/fisiologia , Condrócitos/citologia , Condrócitos/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Proliferação de Células/fisiologia , Tamanho Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Mutantes , Camundongos TransgênicosRESUMO
The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca(2+) release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.
Assuntos
Plasticidade Neuronal/fisiologia , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Animais , Cerebelo/metabolismo , Imunofluorescência , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Desempenho Psicomotor/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.
Assuntos
Clonagem de Organismos/métodos , Neurônios/citologia , Técnicas de Transferência Nuclear , Células de Purkinje/citologia , Animais , Células Cultivadas , Desenvolvimento Embrionário , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desmetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Animais , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismoRESUMO
The N-methyl-D-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.
Assuntos
Encéfalo/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Animais Recém-Nascidos , Técnicas de Introdução de Genes , Camundongos , Transporte Proteico , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Activity-dependent regulation of calcium dynamics in neuronal cells can play significant roles in the modulation of many cellular processes such as intracellular signalling, neuronal activity and synaptic plasticity. Among many calcium influx pathways into neurons, the voltage-dependent calcium channel (VDCC) is the major source of calcium influx, but its modulation by synaptic activity has still been under debate. While the metabotropic glutamate receptor (mGluR) is supposed to modulate L-type VDCCs (L-VDCCs), its reported actions include both facilitation and suppression, probably reflecting the uncertainty of both the molecular targets of the mGluR agonists and the source of the recorded calcium signal in previous reports. In this study, using subtype-specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization-evoked calcium current. This facilitation was not accompanied by the change in single-channel properties of the VDCC itself; instead, it required the activation of calcium-induced calcium release (CICR) that was triggered by VDCC opening, suggesting that the opening of CICR-coupled cation channels was essential for the facilitation. This facilitation was blocked or reduced by the inhibitors of both L-VDCCs and InsP3 receptors (InsP3Rs). Furthermore, L-VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3Rs and L-VDCCs played a pivotal role in the calcium-current facilitation. Finally, we showed that mGluR5 enhanced VDCC-dependent long-term potentiation (LTP) of synaptic transmission. Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity.
Assuntos
Região CA1 Hipocampal/fisiologia , Canais de Cálcio Tipo L/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Células Piramidais/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Glutamato Metabotrópico 5RESUMO
AlkB homolog 1 (ALKBH1) is responsible for the biogenesis of 5-formylcytidine (f5 C) on mitochondrial tRNAMet and essential for mitochondrial protein synthesis. The brain, especially the hippocampus, is highly susceptible to mitochondrial dysfunction; hence, the maintenance of mitochondrial activity is strongly required to prevent disorders associated with hippocampal malfunction. To study the role of ALKBH1 in the hippocampus, we generated dorsal telencephalon-specific Alkbh1 conditional knockout (cKO) mice in inbred C57BL/6 background. These mice showed reduced activity of the respiratory chain complex, hippocampal atrophy, and CA1 pyramidal neuron abnormalities. Furthermore, performances in the fear-conditioning and Morris water maze tests in cKO mice indicated that the hippocampal abnormalities led to impaired hippocampus-dependent learning. These findings indicate critical roles of ALKBH1 in the hippocampus.
Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/deficiência , Região CA1 Hipocampal/metabolismo , Aprendizagem , Células Piramidais/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Animais , Atrofia , Região CA1 Hipocampal/patologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Células Piramidais/patologiaRESUMO
What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that â¼50% of LGI1 and â¼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas ADAM/fisiologia , Encéfalo/metabolismo , Epilepsia/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas do Tecido Nervoso/fisiologia , Proteínas 14-3-3/genética , Animais , Encéfalo/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Subjects with low serum HDL cholesterol levels are reported to be susceptible to diabetes, with insulin resistance believed to be the underlying pathological mechanism. Apolipoprotein M (apoM) is a carrier of sphingosine-1-phosphate (S1P), a multifunctional lipid mediator, on HDL, and the pleiotropic effects of HDL are believed to be mediated by S1P. In the current study, we attempted to investigate the potential association between apoM/S1P and insulin resistance. We observed that the serum levels of apoM were lower in patients with type 2 diabetes and that they were negatively correlated with BMI and the insulin resistance index. While deletion of apoM in mice was associated with worsening of insulin resistance, overexpression of apoM was associated with improvement of insulin resistance. Presumably, apoM/S1P exerts its protective effect against insulin resistance by activating insulin signaling pathways, such as the AKT and AMPK pathways, and also by improving the mitochondrial functions through upregulation of SIRT1 protein levels. These actions of apoM/S1P appear to be mediated via activation of S1P1 and/or S1P3. These results suggest that apoM/S1P exerts protective roles against the development of insulin resistance.
Assuntos
Apolipoproteínas M/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Adulto , Animais , Apolipoproteínas M/genética , Glicemia , Índice de Massa Corporal , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hemoglobinas Glicadas , Células Hep G2 , Humanos , Metabolismo dos Lipídeos , Lipídeos/química , Fígado/química , Lisofosfolipídeos/genética , Masculino , Metaboloma , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Esfingosina/genética , Esfingosina/metabolismoRESUMO
G proteins are posttranslationally modified by isoprenylation: either farnesylation or geranylgeranylation. The gamma subunit of retinal transducin (Talpha/Tbetagamma) is selectively farnesylated, and the farnesylation is required for light signaling mediated by transducin in rod cells. However, whether and how this selective isoprenylation regulates cellular functions remain poorly understood. Here we report that knockin mice expressing geranylgeranylated Tgamma showed normal rod responses to dim flashes under dark-adapted conditions but exhibited impaired properties in light adaptation. Of note, geranylgeranylation of Tgamma suppressed light-induced transition of Tbetagamma from membrane to cytosol, and also attenuated its light-dependent translocation from the outer segment to the inner region, an event contributing to retinal light adaptation. These results indicate that, while the farnesylation of transducin is interchangeable with the geranylgeranylation in terms of the light signaling, the selective farnesylation is important for visual sensitivity regulation by providing sufficient but not excessive membrane anchoring of Tbetagamma.
Assuntos
Adaptação Ocular/fisiologia , Prenilação de Proteína/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo , Visão Ocular/fisiologia , Animais , Membrana Celular/metabolismo , Sensibilidades de Contraste/genética , Glicosilfosfatidilinositóis/metabolismo , Camundongos , Camundongos Transgênicos , Estimulação Luminosa , Subunidades Proteicas/metabolismo , Transporte Proteico/genética , Transducina/genéticaRESUMO
Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Potenciais de Ação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal , Encéfalo/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/patologia , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Células de Purkinje/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Proteína 1 do Complexo Esclerose Tuberosa/deficiência , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/deficiência , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
Rac1 is a member of Rho family GTPases and regulates multiple cellular functions through actin cytoskeleton reorganization. During cerebral corticogenesis, Rac1 has been assumed to be involved in neuronal migration, neurite formation, polarization and axonal guidance. Here we show the specific role of Rac1, regulating midline crossing of commissural axons during cortical development by using cortex-restricted Rac1-knockout mice. In the knockout mice, Rac1 was eliminated from the beginning of corticogenesis exclusively in the dorsal telencephalon where progenitors of cortical projection neurons are located. Cortical lamination was distorted only mildly in the knockout mice, being preserved with six layers of neurons. However, cortex-restricted Rac1 deletion exhibited striking agenesis of commissural axons including the corpus callosum and anterior commissure without affecting other corticofugal axons including corticospinal and corticothalamic projections. Of note, the commissural axons of the knockout mice were potent in extending their process, but failed to cross the midline. Therefore, these findings indicate that Rac1 specifically controls the midline crossing of the commissural fibers, but not axonal formation of corticospinal or corticothalamic fibers during cortical development.
Assuntos
Axônios/fisiologia , Córtex Cerebral/fisiologia , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Transmissão Sináptica/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Axônios/patologia , Encéfalo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Neurônios/patologia , Neuropeptídeos/deficiência , Tratos Piramidais/fisiologia , Telencéfalo/metabolismo , Tálamo/fisiologia , Distribuição Tecidual , Proteínas rac de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTPRESUMO
OBJECTIVE: High-density lipoprotein (HDL) has been epidemiologically shown to be associated with the outcome of sepsis. One potential mechanism is that HDL possesses pleiotropic effects, such as anti-apoptosis, some of which can be ascribed to sphingosine 1-phosphate (S1P) carried on HDL via apolipoprotein M (apoM). Therefore, the aim of this study was to elucidate the roles of apoM/S1P in the consequent lethal conditions of sepsis, such as multiple organ failure caused by severe inflammation and/or disseminated intravascular coagulation. METHODS AND RESULTS: In mice treated with lipopolysaccharide (LPS), both plasma apoM levels and the expression of apoM in the liver and kidney were suppressed. The overexpression of apoM improved the survival rate and ameliorated the elevated plasma alanine aminotransferase (ALT) and creatinine levels, while the knockout or knockdown of apoM deteriorated these parameters in mice treated with LPS. Treatment with VPC23019, an antagonist against S1P receptor 1 and 3, or LY294002, a PI3K inhibitor, partially reversed these protective properties arising from the overexpression of apoM. The overexpression of apoM inhibited the elevation of plasma plasminogen activator inhibitor-1, restored the phosphorylation of Akt, and induced anti-apoptotic changes in the liver, kidney and heart. CONCLUSION: These results suggest that apoM possesses protective properties against LPS-induced organ injuries and could potentially be introduced as a novel therapy for the severe conditions that are consequent to sepsis.
Assuntos
Apolipoproteínas M/metabolismo , Coagulação Intravascular Disseminada/metabolismo , Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Insuficiência de Múltiplos Órgãos/metabolismo , Sepse/metabolismo , Esfingosina/análogos & derivados , Alanina Transaminase/sangue , Animais , Apolipoproteínas M/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Creatinina/sangue , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/imunologia , Lipoproteínas HDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfosserina/análogos & derivados , Fosfosserina/farmacologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/metabolismoRESUMO
Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1ß are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1ß has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering.
RESUMO
We examined the usefulness of commercially available DNA methylation arrays designed for the human genome (Illumina HumanMethylation450 and MethylationEPIC) for high-throughput epigenome analysis of the common marmoset, a nonhuman primate suitable for research on neuropsychiatric disorders. From among the probes on the methylation arrays, we selected those available for the common marmoset. DNA methylation data were obtained from genomic DNA extracted from the frontal cortex and blood samples of adult common marmosets as well as the frontal cortex of neonatal marmosets. About 10% of the probes on the arrays were estimated to be useful for DNA methylation assay in the common marmoset. Strong correlations existed between human and marmoset DNA methylation data. Illumina methylation arrays are useful for epigenome research using the common marmoset.
Assuntos
Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Animais , Callithrix , Epigenômica/instrumentação , Humanos , Masculino , Especificidade da EspécieRESUMO
Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with ß-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.