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Nat Commun ; 10(1): 2588, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197172


The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.

Aneuploidia , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Segregação de Cromossomos/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Instabilidade Genômica , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Cultura Primária de Células , Deleção de Sequência , Fuso Acromático/metabolismo
Am J Pathol ; 188(10): 2392-2405, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30220554


Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.

J Neurosci ; 2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29437890


Transcriptional programs instruct the generation and maintenance of diverse subtypes of neural cells, establishment of distinct brain regions, formation and function of neural circuits, and ultimately behavior. Spatiotemporal and cell type-specific analyses of the transcriptome, the sum total of all RNA transcripts in a cell or an organ, can provide insights into the role of genes in brain development and function, and their potential contribution to disorders of the brain. In the previous decade, advances in sequencing technology and funding from the National Institutes of Health and private foundations for large-scale genomics projects have led to a growing collection of brain transcriptome databases. These valuable resources provide rich and high-quality datasets with spatiotemporal, cell type-specific, and single-cell precision. Most importantly, many of these databases are publicly available via user-friendly web interface, making the information accessible to individual scientists without the need for advanced computational expertise. Here, we highlight key publicly available brain transcriptome databases, summarize the tissue sources and methods used to generate the data, and discuss their utility for neuroscience research.

Invest Ophthalmol Vis Sci ; 54(6): 4007-15, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23640037


PURPOSE: Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties. METHODS: OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system. RESULTS: MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment. CONCLUSIONS: p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema.

Barreira Hematorretiniana/efeitos dos fármacos , Dexametasona/farmacologia , Endotélio Vascular/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Ocludina/biossíntese , Fatores de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Barreira Hematorretiniana/metabolismo , Western Blotting , Bovinos , Células Cultivadas , Imunoprecipitação da Cromatina , Claudina-5/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Endotélio Vascular/metabolismo , Inativação Gênica/fisiologia , Humanos , Masculino , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Fator de Processamento Associado a PTB , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Ratos , Vasos Retinianos/citologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Junções Íntimas/efeitos dos fármacos , Transativadores , Transfecção
Proc Natl Acad Sci U S A ; 109(27): 10855-60, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22711802


Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.

Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Ácidos/química , Calmodulina/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Escherichia coli/genética , Guanilato Quinases/metabolismo , Humanos , Proteína 2 com Domínio MARVEL , Proteínas de Membrana/genética , Mutagênese/fisiologia , Ressonância Magnética Nuclear Biomolecular , Ocludina , Fosfoproteínas/genética , Fosforilação/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Soluções/química , Proteína da Zônula de Oclusão-1
Biochem J ; 446(3): 455-67, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22721706


Pro-inflammatory cytokines and growth factors such as VEGF (vascular endothelial growth factor) contribute to the loss of the BRB (blood-retinal barrier) and subsequent macular oedema in various retinal pathologies. VEGF signalling requires PKCß [conventional PKC (protein kinase C)] activity; however, PKCß inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability, suggesting the involvement of alternative signalling pathways. In the present study, we provide evidence for the involvement of aPKC (atypical PKC) signalling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small-molecule inhibitors, and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small-molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. The results of the present study suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis, and the BBB (blood-brain barrier) in the presence of brain tumours.

Barreira Hematorretiniana/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Retinopatia Diabética/metabolismo , Humanos , Masculino , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/citologia , Retina/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética