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1.
Cell Res ; 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444470

RESUMO

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

2.
Nat Commun ; 10(1): 3615, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399598

RESUMO

Genome-wide association studies have provided evidence for inherited genetic predisposition to chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms underlying CLL risk we analyze chromatin accessibility, active regulatory elements marked by H3K27ac, and DNA methylation at 42 risk loci in up to 486 primary CLLs. We identify that risk loci are significantly enriched for active chromatin in CLL with evidence of being CLL-specific or differentially regulated in normal B-cell development. We then use in situ promoter capture Hi-C, in conjunction with gene expression data to reveal likely target genes of the risk loci. Candidate target genes are enriched for pathways related to B-cell development such as MYC and BCL2 signalling. At 14 loci the analysis highlights 63 variants as the probable functional basis of CLL risk. By integrating genetic and epigenetic information our analysis reveals novel insights into the relationship between inherited predisposition and the regulatory chromatin landscape of CLL.

3.
Cell Metab ; 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31422903

RESUMO

Autophagy facilitates the adaptation to nutritional stress. Here, we show that short-term starvation of cultured cells or mice caused the autophagy-dependent cellular release of acyl-CoA-binding protein (ACBP, also known as diazepam-binding inhibitor, DBI) and consequent ACBP-mediated feedback inhibition of autophagy. Importantly, ACBP levels were elevated in obese patients and reduced in anorexia nervosa. In mice, systemic injection of ACBP protein inhibited autophagy, induced lipogenesis, reduced glycemia, and stimulated appetite as well as weight gain. We designed three approaches to neutralize ACBP, namely, inducible whole-body knockout, systemic administration of neutralizing antibodies, and induction of antiACBP autoantibodies in mice. ACBP neutralization enhanced autophagy, stimulated fatty acid oxidation, inhibited appetite, reduced weight gain in the context of a high-fat diet or leptin deficiency, and accelerated weight loss in response to dietary changes. In conclusion, neutralization of ACBP might constitute a strategy for treating obesity and its co-morbidities.

4.
Biol Psychiatry ; 2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31443933

RESUMO

BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor ß signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor ß signaling and hippocampal function.

5.
Nat Med ; 25(8): 1234-1242, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31332389

RESUMO

The gut microbiome is emerging as a key regulator of several metabolic, immune and neuroendocrine pathways1,2. Gut microbiome deregulation has been implicated in major conditions such as obesity, type 2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease and cancer3-6, but its precise role in aging remains to be elucidated. Here, we find that two different mouse models of progeria are characterized by intestinal dysbiosis with alterations that include an increase in the abundance of Proteobacteria and Cyanobacteria, and a decrease in the abundance of Verrucomicrobia. Consistent with these findings, we found that human progeria patients also display intestinal dysbiosis and that long-lived humans (that is, centenarians) exhibit a substantial increase in Verrucomicrobia and a reduction in Proteobacteria. Fecal microbiota transplantation from wild-type mice enhanced healthspan and lifespan in both progeroid mouse models, and transplantation with the verrucomicrobia Akkermansia muciniphila was sufficient to exert beneficial effects. Moreover, metabolomic analysis of ileal content points to the restoration of secondary bile acids as a possible mechanism for the beneficial effects of reestablishing a healthy microbiome. Our results demonstrate that correction of the accelerated aging-associated intestinal dysbiosis is beneficial, suggesting the existence of a link between aging and the gut microbiota that provides a rationale for microbiome-based interventions against age-related diseases.

6.
Cell Stem Cell ; 25(3): 407-418.e6, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31303548

RESUMO

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

7.
Nat Rev Mol Cell Biol ; 20(7): 385-386, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31164727
8.
J Am Geriatr Soc ; 67(8): 1559-1564, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31045254

RESUMO

Frailty has long been an important concept in the practice of geriatric medicine and in gerontological research, but integration and implementation of frailty concepts into clinical practice in the United States has been slow. The National Institute on Aging (NIA) Intramural Research Program and the Johns Hopkins Older Americans Independence Center sponsored a symposium to identify potential barriers that impede the movement of frailty into clinical practice and to highlight opportunities to facilitate the further integration of frailty into clinical practice. Primary and subspecialty care providers, and investigators working to integrate and translate new biological aging knowledge into more specific preventive and treatment strategies for frailty provided the meeting content. Recommendations included a call for more specific language that clarifies conceptual differences between frailty definitions and measurement tools; the development of randomized controlled trials to test whether specific intervention strategies for a variety of conditions differently affect frail and non-frail individuals; development of implementation studies and therapeutic trials aimed at tailoring care as a function of pragmatic frailty markers; the use of deep learning and dynamic systems approaches to improve the translatability of findings from epidemiological studies; and the incorporation of advances in aging biology, especially focused on mitochondria, stem cells, and senescent cells, toward the further development of biologically targeted intervention and prevention strategies that can be used to treat or prevent frailty. J Am Geriatr Soc 67:1559-1564, 2019.

9.
EMBO Mol Med ; 11(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30862662

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by progerin, a mutant lamin A variant. HGPS patients display accelerated aging and die prematurely, typically from atherosclerosis complications. Recently, we demonstrated that progerin-driven vascular smooth muscle cell (VSMC) loss accelerates atherosclerosis leading to premature death in apolipoprotein E-deficient mice. However, the molecular mechanism underlying this process remains unknown. Using a transcriptomic approach, we identify here endoplasmic reticulum stress (ER) and the unfolded protein responses as drivers of VSMC death in two mouse models of HGPS exhibiting ubiquitous and VSMC-specific progerin expression. This stress pathway was also activated in HGPS patient-derived cells. Targeting ER stress response with a chemical chaperone delayed medial VSMC loss and inhibited atherosclerosis in both progeria models, and extended lifespan in the VSMC-specific model. Our results identify a mechanism underlying cardiovascular disease in HGPS that could be targeted in patients. Moreover, these findings may help to understand other vascular diseases associated with VSMC death, and provide insight into aging-dependent vascular damage related to accumulation of unprocessed toxic forms of lamin A.

18.
Nat Med ; 25(3): 423-426, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778239

RESUMO

CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.


Assuntos
Sistemas CRISPR-Cas , Terapia Genética/métodos , Lamina Tipo A/genética , Progéria/terapia , Animais , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Camundongos , Mutação Puntual , Progéria/genética
19.
J Mol Cell Cardiol ; 128: 38-50, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30625302

RESUMO

RATIONALE: LonP1 is an essential mitochondrial protease, which is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. However, the importance of LonP1 during cardiac stress is largely unknown. OBJECTIVE: To determine the functions of LonP1 during ischemia/reperfusion (I/R) injury in vivo, and hypoxia-reoxygenation (H/R) stress in vitro. METHODS AND RESULTS: LonP1 was induced 2-fold in wild-type mice during cardiac ischemic preconditioning (IPC), which protected the heart against ischemia-reperfusion (I/R) injury. In contrast, haploinsufficiency of LonP1 (LONP1+/-) abrogated IPC-mediated cardioprotection. Furthermore, LONP1+/- mice showed significantly increased infarct size after I/R injury, whereas mice with 3-4 fold cardiac-specific overexpression of LonP1 (LonTg) had substantially smaller infarct size and reduced apoptosis compared to wild-type controls. To investigate the mechanisms underlying cardioprotection, LonTg mice were subjected to ischemia (45 min) followed by short intervals of reperfusion (10, 30, 120 min). During early reperfusion, the left ventricles of LonTg mice showed substantially reduced oxidative protein damage, maintained mitochondrial redox homeostasis, and showed a marked downregulation of both Complex I protein level and activity in contrast to NTg mice. Conversely, when LonP1 was knocked down in isolated neonatal rat ventricular myocytes (NRVMs), an up-regulation of Complex I subunits and electron transport chain (ETC) activities was observed, which was associated with increased superoxide production and reduced respiratory efficiency. The knockdown of LonP1 in NRVMs caused a striking dysmorphology of the mitochondrial inner membrane, mitochondrial hyperpolarization and increased hypoxia-reoxygenation (H/R)-activated apoptosis. Whereas, LonP1 overexpression blocked H/R-induced cell death. CONCLUSIONS: LonP1 is an endogenous mediator of cardioprotection. Our findings show that upregulation of LonP1 mitigates cardiac injury by preventing oxidative damage of proteins and lipids, preserving mitochondrial redox balance and reprogramming bioenergetics by reducing Complex I content and activity. Mechanisms that promote the upregulation of LonP1 could be beneficial in protecting the myocardium from cardiac stress and limiting I/R injury.

20.
Blood ; 133(9): 940-951, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30538135

RESUMO

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1- MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1-/D2- MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1-/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1- MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1- MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1- MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1-/D2-/D3- MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1- MCL.

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