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1.
Nat Commun ; 11(1): 4529, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913184

RESUMO

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.


Assuntos
Metilação de DNA , Epigênese Genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Ilhas de CpG/genética , Estudos Transversais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Técnicas de Introdução de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Carga Global da Doença , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteínas Recombinantes/genética , Sistema de Registros/estatística & dados numéricos , Índice de Gravidade de Doença , Ovinos , Adulto Jovem
2.
Nat Commun ; 11(1): 4581, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917893

RESUMO

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética
3.
Nat Commun ; 11(1): 4855, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978390

RESUMO

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes ß-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.


Assuntos
Astrócitos/metabolismo , Comunicação Celular/fisiologia , Conexina 43/metabolismo , Junções Comunicantes/patologia , Receptores CXCR/metabolismo , Animais , Proliferação de Células , Conexina 43/efeitos dos fármacos , Conexinas/metabolismo , Técnicas de Introdução de Genes , Glioblastoma/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR/agonistas , Receptores CXCR/genética , Transdução de Sinais/fisiologia
4.
Nat Commun ; 11(1): 4337, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859897

RESUMO

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 µM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure.


Assuntos
Reprogramação Celular/fisiologia , Citoplasma/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Sódio/metabolismo , Animais , Modelos Animais de Doenças , Metabolismo Energético , Técnicas de Introdução de Genes , Coração , Hipertrofia , Preparação de Coração Isolado , Masculino , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Sódio/sangue , Trocador de Sódio e Cálcio/efeitos dos fármacos , Tiazepinas/farmacologia
5.
PLoS One ; 15(8): e0238075, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833982

RESUMO

Parkinson disease is the most common neurodegenerative movement disorder, estimated to affect one in twenty-five individuals over the age of 80. Mutations in glucocerebrosidase 1 (GBA1) represent the most common genetic risk factor for Parkinson disease. The link between GBA1 mutations and α-synuclein accumulation, a hallmark of Parkinson disease, is not fully understood. Following our recent finding that Gba1 mutations lead to increased α-synuclein accumulation in mice, we have studied the effects of a single injection of mouse α-synuclein pre-formed fibrils into the striatum of Gba1 mice that carry a L444P knock-in mutation. We found significantly greater formation and spread of α-synuclein inclusions in Gba1-transgenic mice compared to wild-type controls. This indicates that the Gba1 L444P mutation accelerates α-synuclein pathology and spread.


Assuntos
Técnicas de Introdução de Genes , Glucosilceramidase/genética , Mutação , Agregados Proteicos/genética , alfa-Sinucleína/química , Animais , Humanos , Injeções , Camundongos , Neostriado/metabolismo , alfa-Sinucleína/metabolismo
6.
Nat Commun ; 11(1): 3351, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620897

RESUMO

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients.


Assuntos
Proteínas de Transporte/genética , Deficiência Intelectual/genética , Canais Iônicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Transporte/metabolismo , Criança , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Dendritos/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/patologia , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/metabolismo , Cultura Primária de Células , Domínios Proteicos/genética , Índice de Gravidade de Doença , Sequenciamento Completo do Exoma
8.
PLoS One ; 15(7): e0236664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722684

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population. METHODS: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy. RESULTS: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice. CONCLUSION: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.


Assuntos
Anticorpos Antinucleares/genética , Anticorpos Antifosfolipídeos/genética , Antígenos/imunologia , Linfócitos B/imunologia , Anergia Clonal , Lúpus Eritematoso Sistêmico/imunologia , Animais , Linfócitos B/citologia , Proliferação de Células , DNA de Cadeia Simples/imunologia , Suscetibilidade a Doenças , Técnicas de Introdução de Genes , Lúpus Eritematoso Sistêmico/genética , Camundongos
9.
Nat Commun ; 11(1): 2835, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503983

RESUMO

Inherited peripheral neuropathies (IPNs) represent a broad group of disorders including Charcot-Marie-Tooth (CMT) neuropathies characterized by defects primarily arising in myelin, axons, or both. The molecular mechanisms by which mutations in nearly 100 identified IPN/CMT genes lead to neuropathies are poorly understood. Here we show that the Ras-related GTPase Rab35 controls myelin growth via complex formation with the myotubularin-related phosphatidylinositol (PI) 3-phosphatases MTMR13 and MTMR2, encoded by genes responsible for CMT-types 4B2 and B1 in humans, and found that it downregulates lipid-mediated mTORC1 activation, a pathway known to crucially regulate myelin biogenesis. Targeted disruption of Rab35 leads to hyperactivation of mTORC1 signaling caused by elevated levels of PI 3-phosphates and to focal hypermyelination in vivo. Pharmacological inhibition of phosphatidylinositol 3,5-bisphosphate synthesis or mTORC1 signaling ameliorates this phenotype. These findings reveal a crucial role for Rab35-regulated lipid turnover by myotubularins to repress mTORC1 activity and to control myelin growth.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Bainha de Mielina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Astrócitos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Regulação para Baixo , Técnicas de Introdução de Genes , Células HEK293 , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Camundongos Transgênicos , Mutação , Bainha de Mielina/patologia , Cultura Primária de Células , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/genética
10.
Proc Natl Acad Sci U S A ; 117(25): 14532-14542, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513733

RESUMO

Implantation is initiated when an embryo attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed in the endometrium. These events are followed by the formation of an extensive vascular network in the stroma that supports embryonic growth and ensures successful implantation. Interestingly, in many mammalian species, these processes of early pregnancy occur in a hypoxic environment. However, the mechanisms underlying maternal adaptation to hypoxia during early pregnancy remain unclear. In this study, using a knockout mouse model, we show that the transcription factor hypoxia-inducible factor 2 alpha (Hif2α), which is induced in subluminal stromal cells at the time of implantation, plays a crucial role during early pregnancy. Indeed, when preimplantation endometrial stromal cells are exposed to hypoxic conditions in vitro, we observed a striking enhancement in HIF2α expression. Further studies revealed that HIF2α regulates the expression of several metabolic and protein trafficking factors, including RAB27B, at the onset of implantation. RAB27B is a member of the Rab family of GTPases that allows controlled release of secretory granules. These granules are involved in trafficking MMP-9 from the stroma to the epithelium to promote luminal epithelial remodeling during embryo invasion. As pregnancy progresses, the HIF2α-RAB27B pathway additionally mediates crosstalk between stromal and endothelial cells via VEGF granules, developing the vascular network critical for establishing pregnancy. Collectively, our study provides insights into the intercellular communication mechanisms that operate during adaptation to hypoxia, which is essential for embryo implantation and establishment of pregnancy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/fisiologia , Implantação do Embrião/fisiologia , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Comunicação Celular/fisiologia , Linhagem Celular , Embrião de Mamíferos , Endométrio/citologia , Endométrio/metabolismo , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Gravidez , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Células Estromais , Proteínas rab de Ligação ao GTP/genética
11.
Nat Commun ; 11(1): 3133, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561764

RESUMO

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.


Assuntos
Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas Repressoras/genética , Sarcômeros/metabolismo , Animais , Animais Recém-Nascidos , Biotinilação/genética , Feminino , Técnicas de Introdução de Genes , Masculino , Redes e Vias Metabólicas , Camundongos Transgênicos , Modelos Animais , Miocárdio/citologia , Miocárdio/metabolismo , Estudo de Prova de Conceito , Mapas de Interação de Proteínas/fisiologia , Proteínas Quinases/genética , Proteostase/fisiologia , Músculo Quadríceps/citologia , Músculo Quadríceps/metabolismo , Sarcômeros/genética , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
12.
Nat Genet ; 52(7): 719-727, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483291

RESUMO

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.


Assuntos
Regulação da Expressão Gênica , Complexo Mediador/fisiologia , Transcrição Genética , Animais , Linhagem Celular Tumoral , Cromatina/fisiologia , Drosophila , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Complexo Mediador/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo
13.
Nat Immunol ; 21(7): 802-815, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541832

RESUMO

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.


Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Traumatismos do Nervo Facial/patologia , Microglia/metabolismo , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Encéfalo/citologia , Encéfalo/imunologia , Sistemas CRISPR-Cas/genética , Encefalomielite Autoimune Experimental/imunologia , Traumatismos do Nervo Facial/imunologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Microscopia Intravital , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Células NIH 3T3 , RNA-Seq , Análise de Célula Única , Transfecção , Cadeia beta da beta-Hexosaminidase/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-32485164

RESUMO

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Pandemias , Pneumonia Viral , Envelhecimento , Animais , Encéfalo/virologia , Sistemas CRISPR-Cas , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Citocinas/sangue , Técnicas de Introdução de Genes , Pulmão/patologia , Pulmão/virologia , Doenças Pulmonares Intersticiais/patologia , Nariz/virologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/análise , Estômago/virologia , Traqueia/virologia , Carga Viral , Replicação Viral
15.
PLoS Biol ; 18(4): e3000717, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32353004

RESUMO

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/metabolismo
16.
Nat Commun ; 11(1): 2582, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444599

RESUMO

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.


Assuntos
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores Etários , Animais , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Éxons , Feminino , Técnicas de Introdução de Genes , Doença de Huntington/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Fenótipo
17.
Proc Natl Acad Sci U S A ; 117(23): 13105-13116, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457152

RESUMO

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.


Assuntos
Encéfalo/metabolismo , Mapas de Interação de Proteínas/fisiologia , Receptores Opioides delta/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas de Introdução de Genes , Genes Reporter/genética , Masculino , Camundongos , Camundongos Transgênicos , Dobramento de Proteína , Mapeamento de Interação de Proteínas/métodos , Proteômica , Receptores Opioides delta/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem
18.
Neuron ; 107(2): 274-282.e6, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32396852

RESUMO

Single-cell transcriptomics of neocortical neurons have revealed more than 100 clusters corresponding to putative cell types. For inhibitory and subcortical projection neurons (SCPNs), there is a strong concordance between clusters and anatomical descriptions of cell types. In contrast, cortico-cortical projection neurons (CCPNs) separate into surprisingly few transcriptomic clusters, despite their diverse anatomical projection types. We used projection-dependent single-cell transcriptomic analyses and monosynaptic rabies tracing to compare mouse primary visual cortex CCPNs projecting to different higher visual areas. We find that layer 2/3 CCPNs with different anatomical projections differ systematically in their gene expressions, despite forming only a single genetic cluster. Furthermore, these neurons receive feedback selectively from the same areas to which they project. These findings demonstrate that gene-expression analysis in isolation is insufficient to identify neuron types and have important implications for understanding the functional role of cortical feedback circuits.


Assuntos
Neurônios/fisiologia , Animais , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Retroalimentação , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/fisiologia , Rede Nervosa/fisiologia , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/classificação , Vírus da Raiva , Transcriptoma , Córtex Visual/citologia , Córtex Visual/fisiologia
19.
PLoS One ; 15(5): e0233492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469948

RESUMO

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.


Assuntos
Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Polissacarídeos/biossíntese , Polissacarídeos/química , Biotecnologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Manosidases/genética , Manosidases/metabolismo , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Manosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
20.
Nucleic Acids Res ; 48(10): e57, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32232370

RESUMO

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.


Assuntos
Proteínas de Bactérias/metabolismo , Edição de Genes/métodos , Animais , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Feminino , Técnicas de Introdução de Genes , Genômica , Recombinação Homóloga , Humanos , Mutação INDEL , Macaca fascicularis , Camundongos , Ratos Sprague-Dawley , Recombinases Rec A/metabolismo , Peixe-Zebra/genética
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