RESUMEN
Discovery of meningeal lymphatic vessels (LVs) in the dura mater, also known as dural LVs (dLVs) that depend on vascular endothelial growth factor C expression, has raised interest in their possible involvement in Alzheimer's disease (AD). Here we find that in the APdE9 and 5xFAD mouse models of AD, dural amyloid-ß (Aß) is confined to blood vessels and dLV morphology or function is not altered. The induction of sustained dLV atrophy or hyperplasia in the AD mice by blocking or overexpressing vascular endothelial growth factor C, impaired or improved, respectively, macromolecular cerebrospinal fluid (CSF) drainage to cervical lymph nodes. Yet, sustained manipulation of dLVs did not significantly alter the overall brain Aß plaque load. Moreover, dLV atrophy did not alter the behavioral phenotypes of the AD mice, but it improved CSF-to-blood drainage. Our results indicate that sustained dLV manipulation does not affect Aß deposition in the brain and that compensatory mechanisms promote CSF clearance.
RESUMEN
The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II-associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.
Asunto(s)
Vasos Linfáticos , Factor C de Crecimiento Endotelial Vascular , Animales , Ratones , Células Endoteliales/metabolismo , Linfangiogénesis , Enfermedades Neuroinflamatorias , Transducción de Señal , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The meninges, comprising the leptomeninges (pia and arachnoid layers) and the pachymeninx (dura layer), participate in central nervous system (CNS) autoimmunity, but their relative contributions remain unclear. Here we report on findings in animal models of CNS autoimmunity and in patients with multiple sclerosis, where, in acute and chronic disease, the leptomeninges were highly inflamed and showed structural changes, while the dura mater was only marginally affected. Although dural vessels were leakier than leptomeningeal vessels, effector T cells adhered more weakly to the dural endothelium. Furthermore, local antigen-presenting cells presented myelin and neuronal autoantigens less efficiently, and the activation of autoreactive T cells was lower in dural than leptomeningeal layers, preventing local inflammatory processes. Direct antigen application was required to evoke a local inflammatory response in the dura. Together, our data demonstrate an uneven involvement of the meningeal layers in CNS autoimmunity, in which effector T cell trafficking and activation are functionally confined to the leptomeninges, while the dura remains largely excluded from CNS autoimmune processes.
Asunto(s)
Autoinmunidad , Meninges , Esclerosis Múltiple , Animales , Aracnoides , Sistema Nervioso Central , Duramadre , Humanos , Meninges/fisiologíaRESUMEN
Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.
Asunto(s)
Astrocitos/metabolismo , Encefalopatías/genética , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Mitocondrial/metabolismo , Ratones Noqueados , Microscopía Electrónica , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Neuronas/metabolismoRESUMEN
Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
Asunto(s)
Alanina-ARNt Ligasa/genética , Mitocondrias/genética , Edición de ARN , ARN de Transferencia/genética , Aminoacilación de ARN de Transferencia/genética , Alanina-ARNt Ligasa/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Mamíferos , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mutación , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.
Asunto(s)
Vasos Linfáticos/fisiología , Meninges/fisiología , Animales , Animales Recién Nacidos , Transporte Biológico/efectos de los fármacos , Líquido Cefalorraquídeo/metabolismo , Dependovirus/metabolismo , Eliminación de Gen , Humanos , Indoles/farmacología , Inyecciones Intraventriculares , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Masculino , Meninges/efectos de los fármacos , Ratones Endogámicos C57BL , Microesferas , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Transducción de Señal , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiología , Sunitinib , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy.
Asunto(s)
ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Miopatías Mitocondriales/metabolismo , Nucleótidos/metabolismo , Degeneraciones Espinocerebelosas/metabolismo , Adulto , Animales , Carbono/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN Mitocondrial/genética , Femenino , Ácido Fólico/metabolismo , Glucosa/metabolismo , Glutatión/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Mutación , Serina/metabolismo , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/patologíaRESUMEN
ß-catenin has well-established functions in cell growth and differentiation as part of the Wnt signaling pathway and in regulation of cellular adhesion with E-cadherin. Here we studied its significance in midbrain development by temporally controlled deletion of ß-catenin allowing simultaneous analysis of complete (ß-cat-null) and partial (ß-cat-low) loss-of-function phenotypes in progenitor cells. ß-cat-null cells did not contain centrosomes or a microtubule network and were unpolarized forming delaminated bulges. ß-cat-low cells displayed defects in the orientation of the mitotic spindle, increased asymmetric cell divisions and premature differentiation in absence of alterations in polarity or adhesion. The spindle defect was associated with decreased centrosomal S33/S34/T41 phosphorylated ß-catenin (p-ß-cat) and centrosomal and microtubule defects. Interestingly, neural progenitor cells in mice expressing only unphosphorylatable ß-catenin share several phenotypes with ß-catenin loss-of-function mice with defects in microtubules and polarity. The results demonstrate a novel function for p-ß-cat in maintaining neuroepithelial integrity and suggest that centrosomal p-ß-cat is required to maintain symmetric cleavages and polarity in neural progenitors.
Asunto(s)
Centrosoma/metabolismo , Mesencéfalo/embriología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , beta Catenina/metabolismo , Animales , Polaridad Celular/fisiología , Perros , Embrión de Mamíferos/metabolismo , Femenino , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos , Células-Madre Neurales/citología , Neuronas/citología , Fosforilación , beta Catenina/análisisRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with ß-catenin transcriptional activation. Vsnl1 was downregulated in both ß-catenin-stabilized and ß-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised ß-catenin stability, suggesting a counteracting relationship between Vsnl1 and ß-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Neurocalcina/fisiología , Uréter/embriología , Animales , Biomarcadores , Calcio/metabolismo , Ciclo Celular , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , beta Catenina/fisiologíaRESUMEN
beta-Catenin is a multifunctional protein involved in both signalling by secreted factors of Wnt family and regulation of the cellular architecture. We show that beta-catenin stabilization in mouse midbrain-rhombomere 1 region leads to robust up-regulation of several Wnt signalling target genes, including Fgf8. Suggestive of direct transcriptional regulation of the Fgf8 gene, beta-catenin stabilization resulted in Fgf8 up-regulation also in other tissues, specifically in the ventral limb ectoderm. Interestingly, stabilization of beta-catenin rapidly caused down-regulation of the expression of Wnt1 itself, suggesting a negative feedback loop. The changes in signal molecule expression were concomitant with deregulation of anterior-posterior and dorso-ventral patterning. The transcriptional regulatory functions of beta-catenin were confirmed by beta-catenin loss-of-function experiments. Temporally controlled inactivation of beta-catenin revealed a cell-autonomous role for beta-catenin in the maintenance of cell-type specific gene expression in the progenitors of midbrain dopaminergic neurons. These results highlight the role of beta-catenin in establishment of neuroectodermal signalling centers, promoting region-specific gene expression and regulation of cell fate determination.
Asunto(s)
Mesencéfalo/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , beta Catenina/fisiología , Animales , Tipificación del Cuerpo , Factor 8 de Crecimiento de Fibroblastos/genética , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Mesencéfalo/embriología , Ratones , Ratones Transgénicos , Proteína Wnt1/genéticaRESUMEN
Despite recent advances in circadian biology, detailed understanding of how a biological pacemaker system is assembled, maintained, and regulated continues to be a significant challenge. We have assembled and characterized a first-generation, regulatable, self-sustained clock-like expression system based on key components of the mammalian circadian clock. The molecular setup of the clock-like oscillator was reduced to the core set of positive and negative elements common to all known circadian pacemakers. Sophisticated tetracycline-responsive multi-cistronic expression integrated with forefront lentiviral transduction tools enabled autoregulated reporter transgene expression in a human cell line. We characterized transgene expression kinetics of an artificial oscillator and showed that its expression profiles could be modulated by a serum shock and administration of regulating tetracycline antibiotics. Design of a generic mammalian clock-like expression system will offer novel opportunities to study circadian biology and may provide a unique tool for rhythmic expression of desired transgenes fostering advances in biopharmaceutical manufacturing, gene therapy, and tissue engineering.
Asunto(s)
Relojes Biológicos/genética , Ritmo Circadiano/genética , Factores de Transcripción ARNTL , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Relojes Biológicos/fisiología , Western Blotting , Proteínas CLOCK , Proteínas de Ciclo Celular , Línea Celular , Ritmo Circadiano/fisiología , Criptocromos , Retroalimentación Fisiológica/genética , Flavoproteínas/genética , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Genes Reguladores/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Células HeLa , Humanos , Lentivirus/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Circadianas Period , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetraciclina/farmacología , Timidina Quinasa/genética , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , TransfecciónRESUMEN
One of the major challenges in the post-genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation-modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation-inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP-expressing oncoretroviruses to target proliferation-competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd-uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation-modulating genes is essential for developing new strategies for gene therapy and tissue engineering.