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1.
Immunity ; 50(1): 195-211.e10, 2019 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-30635237

RESUMEN

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8+ T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1+PD-1+ TILs mediated the proliferative response to immunotherapy, generating both Tcf1+PD-1+ and differentiated Tcf1-PD-1+ cells. Ablation of Tcf1+PD-1+ TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1+PD-1+ TILs but was essential for the stem-like functions of these cells. Human TCF1+PD-1+ cells were detected among tumor-reactive CD8+ T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular , Proliferación Celular , Receptor 2 Celular del Virus de la Hepatitis A/antagonistas & inhibidores , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Inmunoterapia , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Melanoma/inmunología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL
2.
Immunity ; 45(2): 415-27, 2016 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-27533016

RESUMEN

Chronic infections promote the terminal differentiation (or "exhaustion") of T cells and are thought to preclude the formation of memory T cells. In contrast, we discovered a small subpopulation of virus-specific CD8(+) T cells that sustained the T cell response during chronic infections. These cells were defined by, and depended on, the expression of the transcription factor Tcf1. Transcriptome analysis revealed that this population shared key characteristics of central memory cells but lacked an effector signature. Unlike conventional memory cells, Tcf1-expressing T cells displayed hallmarks of an "exhausted" phenotype, including the expression of inhibitory receptors such as PD-1 and Lag-3. This population was crucial for the T cell expansion that occurred in response to inhibitory receptor blockade during chronic infection. These findings identify a memory-like T cell population that sustains T cell responses and is a prime target for therapeutic interventions to improve the immune response in chronic infections.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Inmunoterapia/métodos , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Factor 1 de Transcripción de Linfocitos T/metabolismo , Adulto , Animales , Antígenos CD/metabolismo , Linfocitos T CD8-positivos/virología , Proliferación Celular , Células Cultivadas , Senescencia Celular , Enfermedad Crónica , Femenino , Humanos , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Transcriptoma , Proteína del Gen 3 de Activación de Linfocitos
3.
Am J Hum Genet ; 108(5): 857-873, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33961779

RESUMEN

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.


Asunto(s)
Encefalopatías/genética , Epilepsia/genética , Riñón Fusionado/genética , Discapacidad Intelectual/genética , Mutación Missense , Proteínas Nucleares/genética , Osteocondrodisplasias/genética , Adolescente , Secuencia de Aminoácidos , Animales , Encefalopatías/etiología , Niño , Preescolar , Epilepsia/complicaciones , Evolución Molecular , Femenino , Frecuencia de los Genes , Humanos , Lactante , Masculino , Ratones , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Fenotipo , Estabilidad Proteica , Síndrome , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Adulto Joven , Pez Cebra/genética
4.
Hum Mol Genet ; 30(19): 1785-1796, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34059922

RESUMEN

Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing (ES) and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant is situated in a loop that might influence the binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner's membrane and in the transient Kolliker's organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining ES, animal modeling, immunohistology and molecular assays.


Asunto(s)
Pérdida Auditiva , Pez Cebra , Animales , Pérdida Auditiva/genética , Humanos , Hidrolasas , Reflejo de Sobresalto , Ubiquitina , Proteasas Ubiquitina-Específicas , Pez Cebra/genética
5.
Am J Physiol Heart Circ Physiol ; 324(4): H504-H518, 2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-36800508

RESUMEN

Upon myocardial infarction (MI), ischemia-induced cell death triggers an inflammatory response responsible for removing necrotic material and inducing tissue repair. TRPM4 is a Ca2+-activated ion channel permeable to monovalent cations. Although its role in cardiomyocyte-driven hypertrophy and arrhythmia post-MI has been established, no study has yet investigated its role in the inflammatory process orchestrated by endothelial cells, immune cells, and fibroblasts. This study aims to assess the role of TRPM4 in 1) survival and cardiac function, 2) inflammation, and 3) healing post-MI. We performed ligation of the left coronary artery or sham intervention on 154 Trpm4 WT or KO mice under isoflurane anesthesia. Survival and echocardiographic functions were monitored up to 5 wk. We collected serum during the acute post-MI phase to analyze proteomes and performed single-cell RNA sequencing on nonmyocytic cells of hearts after 24 and 72 h. Lastly, we assessed chronic fibrosis and angiogenesis. We observed no significant differences in survival or cardiac function, even though our proteomics data showed significantly decreased tissue injury markers (i.e., creatine kinase M and VE-cadherin) in KO serum after 12 h. On the other hand, inflammation, characterized by serum amyloid P component in the serum, higher number of recruited granulocytes, inflammatory monocytes, and macrophages, as well as expression of proinflammatory genes, was significantly higher in KO. This correlated with increased chronic cardiac fibrosis and angiogenesis. Since inflammation and fibrosis are closely linked to adverse remodeling, future therapeutic attempts at inhibiting TRPM4 will need to assess these parameters carefully before proceeding with translational studies.NEW & NOTEWORTHY Deletion of Trpm4 increases markers of cardiac and systemic inflammation within the first 24 h after MI, while inducing an earlier fibrotic transition at 72 h and more overall chronic fibrosis and angiogenesis at 5 wk. The descriptive, robust, and methodologically broad approach of this study sheds light on an important caveat that will need to be taken into account in all future therapeutic attempts to inhibit TRPM4 post-MI.


Asunto(s)
Infarto del Miocardio , Canales Catiónicos TRPM , Ratones , Animales , Células Endoteliales/metabolismo , Multiómica , Miocitos Cardíacos/metabolismo , Inflamación/metabolismo , Fibrosis , Ratones Endogámicos C57BL , Ratones Noqueados , Remodelación Ventricular , Miocardio/metabolismo , Modelos Animales de Enfermedad , Canales Catiónicos TRPM/genética
6.
Glia ; 70(5): 842-857, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34978340

RESUMEN

In amyotrophic lateral sclerosis (ALS) caused by SOD1 gene mutations, both cell-autonomous and noncell-autonomous mechanisms lead to the selective degeneration of motoneurons (MN). Here, we evaluate the therapeutic potential of gene therapy targeting mutated SOD1 in mature astrocytes using mice expressing the mutated SOD1G93A protein. An AAV-gfaABC1 D vector encoding an artificial microRNA is used to deliver RNA interference against mutated SOD1 selectively in astrocytes. The treatment leads to the progressive rescue of neuromuscular junction occupancy, to the recovery of the compound muscle action potential in the gastrocnemius muscle, and significantly improves neuromuscular function. In the spinal cord, gene therapy targeting astrocytes protects a small pool of the most vulnerable fast-fatigable MN until disease end stage. In the gastrocnemius muscle of the treated SOD1G93A mice, the fast-twitch type IIB muscle fibers are preserved from atrophy. Axon collateral sprouting is observed together with muscle fiber type grouping indicative of denervation/reinnervation events. The transcriptome profiling of spinal cord MN shows changes in the expression levels of factors regulating the dynamics of microtubules. Gene therapy delivering RNA interference against mutated SOD1 in astrocytes protects fast-fatigable motor units and thereby improves neuromuscular function in ALS mice.


Asunto(s)
Esclerosis Amiotrófica Lateral , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Animales , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Interferencia de ARN , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética
7.
Kidney Int ; 101(3): 563-573, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34838539

RESUMEN

The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes.


Asunto(s)
Relojes Circadianos , Diabetes Mellitus , Hiperglucemia , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Diabetes Mellitus/metabolismo , Gluconeogénesis , Humanos , Hiperglucemia/metabolismo , Riñón/metabolismo , Túbulos Renales/metabolismo , Ratones
8.
Proc Natl Acad Sci U S A ; 116(34): 17061-17070, 2019 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-31375626

RESUMEN

Hypocretin/orexin (HCRT) and melanin concentrating hormone (MCH) neuropeptides are exclusively produced by the lateral hypothalamus and play important roles in sleep, metabolism, reward, and motivation. Loss of HCRT (ligands or receptors) causes the sleep disorder narcolepsy with cataplexy in humans and in animal models. How these neuropeptides are produced and involved in diverse functions remain unknown. Here, we developed methods to sort and purify HCRT and MCH neurons from the mouse late embryonic hypothalamus. RNA sequencing revealed key factors of fate determination for HCRT (Peg3, Ahr1, Six6, Nr2f2, and Prrx1) and MCH (Lmx1, Gbx2, and Peg3) neurons. Loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers, while knock-down of a Peg3 ortholog in zebrafish completely abolishes their expression, resulting in a 2-fold increase in sleep amount. We also found that loss of HCRT neurons in Hcrt-ataxin-3 mice results in a specific 50% decrease in another orexigenic neuropeptide, QRFP, that might explain the metabolic syndrome in narcolepsy. The transcriptome results were used to develop protocols for the production of HCRT and MCH neurons from induced pluripotent stem cells and ascorbic acid was found necessary for HCRT and BMP7 for MCH cell differentiation. Our results provide a platform to understand the development and expression of HCRT and MCH and their multiple functions in health and disease.


Asunto(s)
Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Melaninas/metabolismo , Neuronas/metabolismo , Orexinas/metabolismo , Hormonas Hipofisarias/metabolismo , Animales , Hormonas Hipotalámicas/genética , Hipotálamo/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melaninas/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Orexinas/genética , Hormonas Hipofisarias/genética
9.
Am J Hum Genet ; 102(1): 116-132, 2018 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-29290337

RESUMEN

Whole-exome and targeted sequencing of 13 individuals from 10 unrelated families with overlapping clinical manifestations identified loss-of-function and missense variants in KIAA1109 allowing delineation of an autosomal-recessive multi-system syndrome, which we suggest to name Alkuraya-Kucinskas syndrome (MIM 617822). Shared phenotypic features representing the cardinal characteristics of this syndrome combine brain atrophy with clubfoot and arthrogryposis. Affected individuals present with cerebral parenchymal underdevelopment, ranging from major cerebral parenchymal thinning with lissencephalic aspect to moderate parenchymal rarefaction, severe to mild ventriculomegaly, cerebellar hypoplasia with brainstem dysgenesis, and cardiac and ophthalmologic anomalies, such as microphthalmia and cataract. Severe loss-of-function cases were incompatible with life, whereas those individuals with milder missense variants presented with severe global developmental delay, syndactyly of 2nd and 3rd toes, and severe muscle hypotonia resulting in incapacity to stand without support. Consistent with a causative role for KIAA1109 loss-of-function/hypomorphic variants in this syndrome, knockdowns of the zebrafish orthologous gene resulted in embryos with hydrocephaly and abnormally curved notochords and overall body shape, whereas published knockouts of the fruit fly and mouse orthologous genes resulted in lethality or severe neurological defects reminiscent of the probands' features.


Asunto(s)
Artrogriposis/genética , Encéfalo/embriología , Mutación/genética , Proteínas/genética , Adolescente , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Linaje , Pez Cebra , Proteínas de Pez Cebra/genética
10.
Rev Med Suisse ; 17(733): 703-707, 2021 Apr 07.
Artículo en Francés | MEDLINE | ID: mdl-33830703

RESUMEN

Compared with the general population, oncology patients face a higher morbidity and mortality caused by the COVID-19 pandemic. As a result, health systems had to quickly adapt cancer care in order to maintain the best quality and patient safety. From March to May and from October to December 2020, 254 patients diagnosed with cancer and tested positive for SARS-CoV-2 benefited from a tele-health monitoring at the Oncology Department at CHUV. This article describes the key points of the development, implementation and operation of this tele-health monitoring, enabled by an interdisciplinary and inter-professional collaboration between different units and healthcare professionals.


En comparaison de la population générale, les patients oncologiques font face à une augmentation de leur morbimortalité en lien avec la pandémie de Covid-19. Par conséquent, les systèmes de santé ont dû s'adapter rapidement dans ce contexte instable afin de poursuivre des soins de qualité tout en assurant la sécurité des patients. De mars à mai ainsi que d'octobre à décembre 2020, un total de 254 patients oncologiques testés positifs au SARS-CoV-2 ont bénéficié d'un suivi téléphonique au Département d'oncologie du CHUV. Cet article décrit les points clés de l'implantation et du fonctionnement de ce télésuivi, grâce à la collaboration entre différentes unités et une équipe interprofessionnelle.


Asunto(s)
COVID-19 , SARS-CoV-2 , Estudios de Seguimiento , Humanos , Pandemias , Teléfono
11.
J Sleep Res ; 28(3): e12718, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-29923248

RESUMEN

Kleine-Levin syndrome (KLS) is a rare periodic hypersomnia with associated behavioural abnormalities but with often favourable prognosis. There is excess risk of KLS in first-degree relatives, suggesting a strong genetic contribution. So far, no mutation is identified in KLS and comprehensive genetic analysis of affected individuals is lacking. Here we performed whole genome single-nucleotide polymorphism (SNP) genotyping and exome sequencing in a large family with seven affected members. The identified gene with a mutation was resequenced in 38 sporadic KLS patients and the expression of the gene product was mapped in the mouse brain. Linkage analysis mapped the disease locus to chromosome 3 and exome analysis identified a heterozygous missense variant in LMOD3 (p.E142D) in the linkage interval. The variant was found to segregate in all affected and one presumably unaffected member of the family. Resequencing LMOD3 in 38 other KLS patients and their families revealed three other low frequency or rare missense variants in seven cases that were inherited with incomplete penetrance. LMOD3 is expressed in the brain and colocalized with major structures involved in the regulation of vigilance states. LMOD proteins are structural proteins and seem to be developmentally regulated. Our findings suggest that KLS might be a structural/neurodevelopmental brain disease.


Asunto(s)
Síndrome de Kleine-Levin/genética , Proteínas de Microfilamentos/genética , Enfermedades del Sistema Nervioso/genética , Adolescente , Adulto , Animales , Encéfalo/metabolismo , Femenino , Humanos , Síndrome de Kleine-Levin/metabolismo , Masculino , Ratones , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Polimorfismo de Nucleótido Simple , Adulto Joven
12.
Brain ; 140(6): 1657-1668, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28460015

RESUMEN

The sleep disorder narcolepsy with cataplexy is characterized by a highly specific loss of hypocretin (orexin) neurons, leading to the hypothesis that the condition is caused by an immune or autoimmune mechanism. All genetic variants associated with narcolepsy are immune-related. Among these are single nucleotide polymorphisms in the P2RY11-EIF3G locus. It is unknown how these genetic variants affect narcolepsy pathogenesis and whether the effect is directly related to P2Y11 signalling or EIF3G function. Exome sequencing in 18 families with at least two affected narcolepsy with cataplexy subjects revealed non-synonymous mutations in the second exon of P2RY11 in two families, and P2RY11 re-sequencing in 250 non-familial cases and 135 healthy control subjects revealed further six different non-synonymous mutations in the second exon of P2RY11 in seven patients. No mutations were found in healthy controls. Six of the eight narcolepsy-associated P2Y11 mutations resulted in significant functional deficits in P2Y11 signalling through both Ca2+ and cAMP signalling pathways. In conclusion, our data show that decreased P2Y11 signalling plays an important role in the development of narcolepsy with cataplexy.


Asunto(s)
Narcolepsia/genética , Narcolepsia/fisiopatología , Receptores Purinérgicos P2/genética , Transducción de Señal/genética , Adulto , Cataplejía/genética , Cataplejía/fisiopatología , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje
13.
J Am Soc Nephrol ; 28(4): 1073-1078, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27799484

RESUMEN

Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.


Asunto(s)
Síndrome de Fanconi/etiología , Nefronas , Receptores Acoplados a Proteínas G/fisiología , Receptores Virales/fisiología , Raquitismo Hipofosfatémico/etiología , Animales , Femenino , Masculino , Ratones , Receptor de Retrovirus Xenotrópico y Politrópico
14.
Plant Physiol ; 170(1): 385-400, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26546667

RESUMEN

The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatos/metabolismo , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Transducción de Señal , Nicotiana/genética , Red trans-Golgi/metabolismo
15.
J Am Soc Nephrol ; 27(10): 2997-3004, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27056296

RESUMEN

The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1lox/lox/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD+-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.


Asunto(s)
Factores de Transcripción ARNTL/genética , Relojes Circadianos/genética , Diuréticos/metabolismo , Furosemida/metabolismo , Riñón/metabolismo , Metaboloma/genética , Animales , Diuréticos/sangre , Furosemida/sangre , Ratones , Nefronas
16.
Am J Hum Genet ; 92(6): 990-5, 2013 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-23684011

RESUMEN

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.


Asunto(s)
Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Anomalías Craneofaciales/genética , Enanismo/genética , Hiperostosis Cortical Congénita/genética , Hipocalcemia/genética , Hipoparatiroidismo/genética , Receptores Virales/genética , Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/mortalidad , Anomalías Múltiples/patología , Adolescente , Adulto , Enfermedades del Desarrollo Óseo/mortalidad , Enfermedades del Desarrollo Óseo/patología , Niño , Anomalías Craneofaciales/mortalidad , Anomalías Craneofaciales/patología , Enanismo/diagnóstico por imagen , Enanismo/mortalidad , Estudios de Asociación Genética , Heterocigoto , Humanos , Hiperostosis Cortical Congénita/diagnóstico por imagen , Hiperostosis Cortical Congénita/mortalidad , Hipocalcemia/diagnóstico por imagen , Hipocalcemia/mortalidad , Hipoparatiroidismo/diagnóstico por imagen , Hipoparatiroidismo/mortalidad , Lactante , Recién Nacido , Masculino , Mutación Missense , Hormona Paratiroidea/deficiencia , Radiografía
17.
Biometals ; 28(3): 521-8, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25753945

RESUMEN

Approximately 0.2% of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1% zinc, >0.1% nickel or >0.01% cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.


Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/parasitología , Cadmio/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/parasitología , Animales , Mariposas Diurnas/patogenicidad , Regulación de la Expresión Génica de las Plantas , Herbivoria , Larva/patogenicidad
18.
PLoS Genet ; 8(4): e1002652, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22511887

RESUMEN

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.


Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ARN Polimerasa II , ARN Mensajero , Arabidopsis/genética , Flores/genética , Flores/crecimiento & desarrollo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Meiosis/genética , Mutación , Proteínas Nucleares/genética , Fenotipo , Plantas Modificadas Genéticamente , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie
19.
J Am Soc Nephrol ; 25(7): 1430-9, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24652800

RESUMEN

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1(lox/lox)/Ren1(d)Cre mice). Analysis of Bmal1(lox/lox)/Ren1(d)Cre mice showed that the floxed Bmal1 allele was excised in the kidney. In the kidney, BMAL1 protein expression was absent in the renin-secreting granular cells of the juxtaglomerular apparatus and the collecting duct. A partial reduction of BMAL1 expression was observed in the medullary thick ascending limb. Functional analyses showed that Bmal1(lox/lox)/Ren1(d)Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis.


Asunto(s)
Presión Sanguínea/fisiología , Relojes Circadianos/fisiología , Homeostasis/fisiología , Equilibrio Hidroelectrolítico/fisiología , Factores de Transcripción ARNTL/fisiología , Animales , Masculino , Ratones , Renina/fisiología
20.
J Neurosci ; 33(12): 5127-37, 2013 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-23516279

RESUMEN

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.


Asunto(s)
MicroARNs/fisiología , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Transcriptoma , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/fisiología , Diferenciación Celular/fisiología , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Femenino , Masculino , MicroARNs/genética , Microglía/citología , Microglía/fisiología , Células-Madre Neurales/citología , Neuroglía/citología , Neuronas/citología , Oligodendroglía/citología , Oligodendroglía/fisiología , Fenotipo , Cultivo Primario de Células , Ratas , Sinapsis/fisiología
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