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1.
Nat Commun ; 12(1): 4722, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354059

RESUMO

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.


Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Dependovirus/genética , Feminino , Humanos , Lamina Tipo A/deficiência , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução Genética
2.
Nat Commun ; 12(1): 4825, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376658

RESUMO

Circular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.


Assuntos
Encéfalo/metabolismo , Éxons/genética , Íntrons/genética , Sequenciamento por Nanoporos/métodos , RNA Circular/genética , Análise de Sequência de RNA/métodos , Animais , Expressão Gênica , Humanos , Masculino , Camundongos da Linhagem 129 , Isoformas de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445339

RESUMO

Both agonist studies and loss-of-function models indicate that PPARγ plays an important role in cutaneous biology. Since PPARγ has a high level of basal activity, we hypothesized that epidermal PPARγ would regulate normal homeostatic processes within the epidermis. In this current study, we performed mRNA sequencing and differential expression analysis of epidermal scrapings from knockout mice and wildtype littermates. Pparg-/-epi mice exhibited a 1.5-fold or greater change in the expression of 11.8% of 14,482 identified transcripts. Up-regulated transcripts included those for a large number of cytokines/chemokines and their receptors, as well as genes associated with inflammasome activation and keratinization. Several of the most dramatically up-regulated pro-inflammatory genes in Pparg-/-epi mouse skin included Igfl3, 2610528A11Rik, and Il1f6. RT-PCR was performed from RNA obtained from non-lesional full-thickness skin and verified a marked increase in these transcripts, as well as transcripts for Igflr1, which encodes the receptor for Igfl3, and the 2610528A11Rik receptor (Gpr15). Transcripts for Il4 were detected in Pparg-/-epi mouse skin, but transcripts for Il17 and Il22 were not detected. Down-regulated transcripts included sebaceous gland markers and a number of genes associated with lipid barrier formation. The change in these transcripts correlates with an asebia phenotype, increased transepidermal water loss, alopecia, dandruff, and the appearance of spontaneous inflammatory skin lesions. Histologically, non-lesional skin showed hyperkeratosis, while inflammatory lesions were characterized by dermal inflammation and epidermal acanthosis, spongiosis, and parakeratosis. In conclusion, loss of epidermal Pparg alters a substantial set of genes that are associated with cutaneous inflammation, keratinization, and sebaceous gland function. The data indicate that epidermal PPARγ plays an important role in homeostatic epidermal function, particularly epidermal differentiation, barrier function, sebaceous gland development and function, and inflammatory signaling.


Assuntos
Dermatite/genética , Epiderme/metabolismo , PPAR gama/fisiologia , Fenômenos Fisiológicos da Pele/genética , Animais , Células Cultivadas , Dermatite/metabolismo , Dermatite/patologia , Dermatite/fisiopatologia , Epiderme/fisiologia , Homeostase/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , PPAR gama/genética , PPAR gama/metabolismo
4.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445342

RESUMO

Epigenetic regulation by histone deacetylase (HDAC) is associated with synaptic plasticity and memory formation, and its aberrant expression has been linked to cognitive disorders, including Alzheimer's disease (AD). This study aimed to investigate the role of class IIa HDAC expression in AD and monitor it in vivo using a novel radiotracer, 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]TFAHA). A human neural cell culture model with familial AD (FAD) mutations was established and used for in vitro assays. Positron emission tomography (PET) imaging with [18F]TFAHA was performed in a 3xTg AD mouse model for in vivo evaluation. The results showed a significant increase in HDAC4 expression in response to amyloid-ß (Aß) deposition in the cell model. Moreover, treatment with an HDAC4 selective inhibitor significantly upregulated the expression of neuronal memory-/synaptic plasticity-related genes. In [18F]TFAHA-PET imaging, whole brain or regional uptake was significantly higher in 3xTg AD mice compared with WT mice at 8 and 11 months of age. Our study demonstrated a correlation between class IIa HDACs and Aßs, the therapeutic benefit of a selective inhibitor, and the potential of using [18F]TFAHA as an epigenetic radiotracer for AD, which might facilitate the development of AD-related neuroimaging approaches and therapies.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/fisiologia , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacocinética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Histona Desacetilases/classificação , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroimagem/métodos , Tomografia por Emissão de Pósitrons/métodos , Células Tumorais Cultivadas
5.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445681

RESUMO

Parthenogenetic embryos have been widely studied as an effective tool related to paternal and maternal imprinting genes and reproductive problems for a long time. In this study, we established a parthenogenetic epiblast-like stem cell line through culturing parthenogenetic diploid blastocysts in a chemically defined medium containing activin A and bFGF named paAFSCs. The paAFSCs expressed pluripotent marker genes and germ-layer-related genes, as well as being alkaline-phosphatase-positive, which is similar to epiblast stem cells (EpiSCs). We previously showed that advanced embryonic stem cells (ASCs) represent hypermethylated naive pluripotent embryonic stem cells (ESCs). Here, we converted paAFSCs to ASCs by replacing bFGF with bone morphogenetic protein 4 (BMP4), CHIR99021, and leukemia inhibitory factor (LIF) in a culture medium, and we obtained parthenogenetic advanced stem cells (paASCs). The paASCs showed similar morphology with ESCs and also displayed a stronger developmental potential than paAFSCs in vivo by producing chimaeras. Our study demonstrates that maternal genes could support parthenogenetic EpiSCs derived from blastocysts and also have the potential to convert primed state paAFSCs to naive state paASCs.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Partenogênese/fisiologia , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Técnicas de Cultura Embrionária/métodos , Feminino , Fatores de Crescimento de Fibroblastos/farmacologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/citologia , Partenogênese/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia
6.
Nat Commun ; 12(1): 4662, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341345

RESUMO

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Nefropatias Diabéticas/metabolismo , Compostos Orgânicos/farmacologia , Podócitos/metabolismo , Proteinúria/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Knockout , Estrutura Molecular , Niacinamida/química , Niacinamida/farmacologia , Compostos Orgânicos/síntese química , Compostos Orgânicos/química , Podócitos/citologia , Interferência de RNA , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Células THP-1
7.
Neuron ; 109(18): 2864-2883.e8, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34384519

RESUMO

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.


Assuntos
Proteínas do Domínio Armadillo/biossíntese , Axônios/metabolismo , Proteínas do Citoesqueleto/biossíntese , Proteínas de Drosophila/biossíntese , Evolução Molecular , Morfogênese/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Proteínas do Domínio Armadillo/antagonistas & inibidores , Proteínas do Domínio Armadillo/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Gravidez , Proteínas Serina-Treonina Quinases/genética
8.
Elife ; 102021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34423776

RESUMO

While recent studies have uncovered dedicated neural pathways mediating the positive control of parenting, the regulation of infant-directed aggression and how it relates to adult-adult aggression is poorly understood. Here we show that urocortin-3 (Ucn3)-expressing neurons in the hypothalamic perifornical area (PeFAUcn3) are activated during infant-directed attacks in males and females, but not other behaviors. Functional manipulations of PeFAUcn3 neurons demonstrate the role of this population in the negative control of parenting in both sexes. PeFAUcn3 neurons receive input from areas associated with vomeronasal sensing, stress, and parenting, and send projections to hypothalamic and limbic areas. Optogenetic activation of PeFAUcn3 axon terminals in these regions triggers various aspects of infant-directed agonistic responses, such as neglect, repulsion, and aggression. Thus, PeFAUcn3 neurons emerge as a dedicated circuit component controlling infant-directed neglect and aggression, providing a new framework to understand the positive and negative regulation of parenting in health and disease.


Assuntos
Agressão , Comportamento Animal , Hipotálamo/metabolismo , Comportamento Materno , Neurônios/metabolismo , Comportamento Paterno , Urocortinas/metabolismo , Animais , Feminino , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/metabolismo , Optogenética , Fatores Sexuais , Urocortinas/genética
9.
Arterioscler Thromb Vasc Biol ; 41(9): 2483-2493, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34320838

RESUMO

Objective: Despite considerable research, the goal of finding nonsurgical remedies against thoracic aortic aneurysm and acute aortic dissection remains elusive. We sought to identify a novel aortic PK (protein kinase) that can be pharmacologically targeted to mitigate aneurysmal disease in a well-established mouse model of early-onset progressively severe Marfan syndrome (MFS). Approach and Results: Computational analyses of transcriptomic data derived from the ascending aorta of MFS mice predicted a probable association between thoracic aortic aneurysm and acute aortic dissection development and the multifunctional, stress-activated HIPK2 (homeodomain-interacting protein kinase 2). Consistent with this prediction, Hipk2 gene inactivation significantly extended the survival of MFS mice by slowing aneurysm growth and delaying transmural rupture. HIPK2 also ranked among the top predicted PKs in computational analyses of DEGs (differentially expressed genes) in the dilated aorta of 3 MFS patients, which strengthened the clinical relevance of the experimental finding. Additional in silico analyses of the human and mouse data sets identified the TGF (transforming growth factor)-ß/Smad3 signaling pathway as a potential target of HIPK2 in the MFS aorta. Chronic treatment of MFS mice with an allosteric inhibitor of HIPK2-mediated stimulation of Smad3 signaling validated this prediction by mitigating thoracic aortic aneurysm and acute aortic dissection pathology and partially improving aortic material stiffness. Conclusions: HIPK2 is a previously unrecognized determinant of aneurysmal disease and an attractive new target for antithoracic aortic aneurysm and acute aortic dissection multidrug therapy.


Assuntos
Aneurisma Dissecante/prevenção & controle , Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Fibrilina-1/genética , Síndrome de Marfan/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Remodelação Vascular/efeitos dos fármacos , Adulto , Aneurisma Dissecante/enzimologia , Aneurisma Dissecante/genética , Aneurisma Dissecante/patologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/enzimologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Síndrome de Marfan/complicações , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Proteína Smad3/metabolismo
10.
Cell Death Dis ; 12(7): 700, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262022

RESUMO

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation, which is controlled by genetic, epigenetic, and epitranscriptomic factors. Here we generate RNA demethylase Fto and methyltransferase Mettl3 cortical-specific conditional knockout mice, and detect severe brain defects caused by Mettl3 deletion but not Fto knockout. Transcriptomic profiles using RNA sequencing indicate that knockout of Mettl3 causes a more dramatic alteration on gene transcription than that of Fto. Interestingly, we conduct ribosome profiling sequencing, and find that knockout of Mettl3 leads to a more severe disruption of translational regulation of mRNAs than deletion of Fto and results in altered translation of crucial genes in cortical radial glial cells and intermediate progenitors. Moreover, Mettl3 deletion causes elevated translation of a significant number of mRNAs, in particular major components in m6A methylation. Our findings indicate distinct functions of Mettl3 and Fto in brain development, and uncover a profound role of Mettl3 in regulating translation of major mRNAs that control proper cortical development.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Córtex Cerebral/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Metiltransferases/metabolismo , Biossíntese de Proteínas , Transcrição Genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Córtex Cerebral/embriologia , Idade Gestacional , Metilação , Metiltransferases/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurogênese , Neuroglia/enzimologia , Neuroglia/patologia , Processamento Pós-Transcricional do RNA , Transcriptoma
11.
Nat Commun ; 12(1): 3370, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099647

RESUMO

A sensitive approach to quantitative analysis of transcriptional regulation in diploid organisms is analysis of allelic imbalance (AI) in RNA sequencing (RNA-seq) data. A near-universal practice in such studies is to prepare and sequence only one library per RNA sample. We present theoretical and experimental evidence that data from a single RNA-seq library is insufficient for reliable quantification of the contribution of technical noise to the observed AI signal; consequently, reliance on one-replicate experimental design can lead to unaccounted-for variation in error rates in allele-specific analysis. We develop a computational approach, Qllelic, that accurately accounts for technical noise by making use of replicate RNA-seq libraries. Testing on new and existing datasets shows that application of Qllelic greatly decreases false positive rate in allele-specific analysis while conserving appropriate signal, and thus greatly improves reproducibility of AI estimates. We explore sources of technical overdispersion in observed AI signal and conclude by discussing design of RNA-seq studies addressing two biologically important questions: quantification of transcriptome-wide AI in one sample, and differential analysis of allele-specific expression between samples.


Assuntos
Desequilíbrio Alélico , Biblioteca Gênica , Polimorfismo de Nucleotídeo Único , RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Algoritmos , Alelos , Animais , Feminino , Camundongos da Linhagem 129 , Modelos Genéticos , RNA/metabolismo
12.
Cell Death Dis ; 12(7): 625, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135312

RESUMO

Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Clobetasol/farmacologia , Glucocorticoides/farmacologia , Proteínas Hedgehog/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/inervação , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Coluna Vertebral/efeitos dos fármacos , Esclerose Amiotrófica Lateral/induzido quimicamente , Esclerose Amiotrófica Lateral/imunologia , Esclerose Amiotrófica Lateral/metabolismo , Animais , Estudos de Casos e Controles , Toxina da Cólera , Bases de Dados Genéticas , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos da Linhagem 129 , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Neurônios Motores/imunologia , Neurônios Motores/metabolismo , Teste de Campo Aberto , Saporinas , Transdução de Sinais , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Coluna Vertebral/imunologia , Coluna Vertebral/metabolismo , Coluna Vertebral/fisiopatologia
13.
Elife ; 102021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096504

RESUMO

Spatially distributed excitation and inhibition collectively shape a visual neuron's receptive field (RF) properties. In the direction-selective circuit of the mammalian retina, the role of strong null-direction inhibition of On-Off direction-selective ganglion cells (On-Off DSGCs) on their direction selectivity is well-studied. However, how excitatory inputs influence the On-Off DSGC's visual response is underexplored. Here, we report that On-Off DSGCs have a spatially displaced glutamatergic receptive field along their horizontal preferred-null motion axes. This displaced receptive field contributes to DSGC null-direction spiking during interrupted motion trajectories. Theoretical analyses indicate that population responses during interrupted motion may help populations of On-Off DSGCs signal the spatial location of moving objects in complex, naturalistic visual environments. Our study highlights that the direction-selective circuit exploits separate sets of mechanisms under different stimulus conditions, and these mechanisms may help encode multiple visual features.


Assuntos
Potenciais Evocados Visuais , Potenciais Pós-Sinápticos Excitadores , Percepção de Movimento , Células Ganglionares da Retina/fisiologia , Transmissão Sináptica , Campos Visuais , Animais , Sinalização do Cálcio , Feminino , Ácido Glutâmico/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Estimulação Luminosa , Células Ganglionares da Retina/metabolismo , Fatores de Tempo
14.
Neurology ; 96(18): e2332-e2345, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34032594

RESUMO

OBJECTIVE: To develop a reliable and fast assay to quantify the α-synuclein (α-syn)-containing extracellular vesicles (EVs) in CSF and to assess their diagnostic potential for Parkinson disease (PD). METHODS: A cross-sectional, multicenter study was designed, including 170 patients with PD and 131 healthy controls (HCs) with a similar distribution of age and sex recruited from existing center studies at the University of Washington and Oregon Health and Science University. CSF EVs carrying α-syn or aggregated α-syn were quantified using antibodies against total or aggregated α-syn, respectively, and highly specific, sensitive, and rapid assays based on the novel Apogee nanoscale flow cytometry technology. RESULTS: No significant differences in the number and size distribution of total EVs between patients with PD and HCs in CSF were observed. When examining the total α-syn-positive and aggregated α-syn-positive EV subpopulations, the proportions of both among all detected CSF EVs were significantly lower in patients with PD compared to HCs (p < 0.0001). While each EV subpopulation showed better diagnostic sensitivity and specificity than total CSF α-syn measured directly with an immunoassay, a combination of the 2 EV subpopulations demonstrated a diagnostic accuracy that attained clinical relevance (area under curve 0.819, sensitivity 80%, specificity 71%). CONCLUSION: Using newly established, sensitive nanoscale flow cytometry assays, we have demonstrated that total α-syn-positive and aggregated α-syn-positive EVs in CSF may serve as a helpful tool in PD diagnosis. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that total and aggregated α-syn-positive EVs in CSF identify patients with PD.


Assuntos
Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Nanotecnologia/métodos , Doença de Parkinson/diagnóstico , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Animais , Estudos Transversais , Vesículas Extracelulares/química , Feminino , Humanos , Imunoensaio/métodos , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , alfa-Sinucleína/análise
15.
Cell Death Dis ; 12(6): 521, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021120

RESUMO

The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues. The canonical Wnt pathway signals MPCs to commit to osteogenic differentiation at the expense of adipogenic fate. In contrast to ß-catenin, p53's anti-osteogenic function is much less understood. Both activities are thought to be achieved through targeting Runx2 and/or Osterix (Osx, Sp7) transcription. Precisely, how Osx activity is dictated by ß-catenin or p53 is not clarified and represents a knowledge gap that, until now, has largely been taken for granted. Using conditional lineage-tracing mice, we demonstrated that chondrocytes gave rise to a sizable fraction of MPCs, which served as progenitors of chondrocyte-derived osteoblasts (Chon-ob). Wnt/ß-catenin activity was only required at the stage of chondrocyte-derived mesenchymal progenitor (C-MPC) to Chon-ob differentiation. ß-catenin- C-MPCs lost osteogenic ability and favored adipogenesis. Mechanistically, we discovered that p53 activity was elevated in ß-catenin- MPCs including ß-catenin- C-MPCs and deleting p53 from the ß-catenin- MPCs fully restored osteogenesis. While high levels of p53 were present in the nuclei of ß-catenin- MPCs, Osx was confined to the cytoplasm, implying a mechanism that did not involve direct p53-Osx interaction. Furthermore, we found that p53's anti-osteogenic activity was dependent on its DNA-binding ability. Our findings identify chondrocytes as an additional source for MPCs and indicate that Wnt/ß-catenin discretely regulates chondrocyte to C-MPC and the subsequent C-MPC to osteoblast developments. Most of all we unveil a previously unrecognized functional link between ß-catenin and p53, placing p53's negative role in the context of Wnt/ß-catenin signaling-induced MPC osteogenic differentiation.


Assuntos
Células-Tronco Mesenquimais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt/fisiologia , Adipogenia/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/genética , Regulação para Baixo/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/fisiologia , Osteogênese/genética , Proteína Supressora de Tumor p53/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
16.
Cell Death Dis ; 12(6): 525, 2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34023861

RESUMO

While pregnancy is known to reduce a woman's life-long risk of breast cancer, clinical data suggest that it can specifically promote HER2 (human EGF receptor 2)-positive breast cancer subtype (HER2+ BC). HER2+ BC, characterized by amplification of HER2, comprises about 20% of all sporadic breast cancers and is more aggressive than hormone receptor-positive breast cancer (the majority of cases). Consistently with human data, pregnancy strongly promotes HER2+ BC in genetic mouse models. One proposed mechanism of this is post-pregnancy accumulation of PIMECs (pregnancy-identified mammary epithelial cells), tumor-initiating cells for HER2+ BC in mice. We previously showed that p63, a homologue of the tumor suppressor p53, is required to maintain the post-pregnancy number of PIMECs and thereby promotes HER2+ BC. Here we set to test whether p63 also affects the intrinsic tumorigenic properties of PIMECs. To this end, we FACS-sorted YFP-labeled PIMECs from p63+/-;ErbB2 and control p63+/+;ErbB2 females and injected their equal amounts into immunodeficient recipients. To our surprise, p63+/- PIMECs showed increased, rather than decreased, tumorigenic capacity in vivo, i.e., significantly accelerated tumor onset and tumor growth, as well as increased self-renewal in mammosphere assays and proliferation in vitro and in vivo. The underlying mechanism of these phenotypes seems to be a specific reduction of the tumor suppressor TAp63 isoform in p63+/- luminal cells, including PIMECs, with concomitant aberrant upregulation of the oncogenic ΔNp63 isoform, as determined by qRT-PCR and scRNA-seq analyses. In addition, scRNA-seq revealed upregulation of several cancer-associated (Il-4/Il-13, Hsf1/HSP), oncogenic (TGFß, NGF, FGF, MAPK) and self-renewal (Wnt, Notch) pathways in p63+/-;ErbB2 luminal cells and PIMECs per se. Altogether, these data reveal a complex role of p63 in PIMECs and pregnancy-associated HER2+ BC: maintaining the amount of PIMECs while suppressing their intrinsic tumorigenic capacity.


Assuntos
Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/fisiologia , Gravidez/fisiologia , Transativadores/fisiologia , Animais , Mama/patologia , Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Feminino , Genes erbB-2 , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Células-Tronco Neoplásicas/patologia , Transativadores/genética
17.
Exp Neurol ; 342: 113755, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33984337

RESUMO

BACKGROUND: RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death in in vitro and in vivo models of Parkinson's and Huntington's diseases and is up regulated in compromised neurons in human postmortem brains of both neurodegenerative disorders. Indeed, in both Parkinson's and Huntington's disease mouse models, RTP801 knockdown alleviates motor-learning deficits. RESULTS: We investigated the physiological role of RTP801 in neuronal plasticity and we found RTP801 in rat, mouse and human synapses. The absence of RTP801 enhanced excitatory synaptic transmission in both neuronal cultures and brain slices from RTP801 knock-out (KO) mice. Indeed, RTP801 KO mice showed improved motor learning, which correlated with lower spine density but increased basal filopodia and mushroom spines in the motor cortex layer V. This paralleled with higher levels of synaptosomal GluA1 and TrkB receptors in homogenates derived from KO mice motor cortex, proteins that are associated with synaptic strengthening. CONCLUSIONS: Altogether, these results indicate that RTP801 has an important role modulating neuronal plasticity and motor learning. They will help to understand its role in neurodegenerative disorders where RTP801 levels are detrimentally upregulated.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Aprendizagem/fisiologia , Córtex Motor/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Sinapses/genética
18.
Front Immunol ; 12: 661290, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995384

RESUMO

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.


Assuntos
Colo/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Fagócitos/imunologia , Células Th17/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígeno CD24/imunologia , Antígeno CD24/metabolismo , Colo/citologia , Colo/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Expressão Gênica/imunologia , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Intestino Delgado/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Fagócitos/metabolismo , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Células Th17/metabolismo
19.
Elife ; 102021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34013884

RESUMO

Glutamatergic neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating breathing in response to tissue CO2/H+. The RTN and greater parafacial region may also function as a chemosensing network composed of CO2/H+-sensitive excitatory and inhibitory synaptic interactions. In the context of disease, we showed that loss of inhibitory neural activity in a mouse model of Dravet syndrome disinhibited RTN chemoreceptors and destabilized breathing (Kuo et al., 2019). Despite this, contributions of parafacial inhibitory neurons to control of breathing are unknown, and synaptic properties of RTN neurons have not been characterized. Here, we show the parafacial region contains a limited diversity of inhibitory neurons including somatostatin (Sst)-, parvalbumin (Pvalb)-, and cholecystokinin (Cck)-expressing neurons. Of these, Sst-expressing interneurons appear uniquely inhibited by CO2/H+. We also show RTN chemoreceptors receive inhibitory input that is withdrawn in a CO2/H+-dependent manner, and chemogenetic suppression of Sst+ parafacial neurons, but not Pvalb+ or Cck+ neurons, increases baseline breathing. These results suggest Sst-expressing parafacial neurons contribute to RTN chemoreception and respiratory activity.


Assuntos
Dióxido de Carbono/metabolismo , Células Quimiorreceptoras/metabolismo , Epilepsias Mioclônicas/metabolismo , Hidrogênio/metabolismo , Núcleos Intralaminares do Tálamo/metabolismo , Pulmão/inervação , Respiração , Somatostatina/metabolismo , Animais , Modelos Animais de Doenças , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/fisiopatologia , Feminino , Ácido Glutâmico/metabolismo , Núcleos Intralaminares do Tálamo/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Inibição Neural , Somatostatina/genética , Transmissão Sináptica
20.
Elife ; 102021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34036937

RESUMO

Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-ß, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.


Assuntos
Tendão do Calcâneo/patologia , Proteínas da Matriz Extracelular/deficiência , Atividade Motora , Osteogênese Imperfeita/patologia , Osteogênese Imperfeita/fisiopatologia , Ligamento Patelar/patologia , Tendão do Calcâneo/metabolismo , Actinas/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Genes Recessivos , Predisposição Genética para Doença , Força da Mão , Metaloproteinase 2 da Matriz/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , NF-kappa B/metabolismo , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Ligamento Patelar/metabolismo , Fenótipo , Fosforilação , Resistência Física , Células-Tronco/metabolismo , Células-Tronco/patologia
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