RESUMEN
Raising reactive oxygen species (ROS) levels in cancer cells to cause macromolecular damage and cell death is a promising anticancer treatment strategy. Observations that electromagnetic fields (EMF) elevate intracellular ROS and cause cancer cell death, have led us to develop a new portable wearable EMF device that generates spinning oscillating magnetic fields (sOMF) to selectively kill cancer cells while sparing normal cells in vitro and to shrink GBM tumors in vivo through a novel mechanism. Here, we characterized the precise configurations and timings of sOMF stimulation that produce cytotoxicity due to a critical rise in superoxide in two types of human glioma cells. We also found that the antioxidant Trolox reverses the cytotoxic effect of sOMF on glioma cells indicating that ROS play a causal role in producing the effect. Our findings clarify the link between the physics of magnetic stimulation and its mechanism of anticancer action, facilitating the development of a potential new safe noninvasive device-based treatment for GBM and other gliomas.
Asunto(s)
Glioma , Estrés Oxidativo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Glioma/terapia , Glioma/patología , Superóxidos , Campos ElectromagnéticosRESUMEN
Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult CNS results in region-specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiologic analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis.SIGNIFICANCE STATEMENT Mutations in Mediator protein complex subunit 12 (Med12) are associated with X-linked intellectual disability syndromes and hearing loss. Using temporal-conditional genetic approaches in CNS glia, we found that loss of Med12 results in severe hearing loss in adult animals through rapid degeneration of the stria vascularis. Our study describes the first animal model that recapitulates hearing loss identified in Med12-related disorders and provides a new system in which to examine the underlying cellular and molecular mechanisms of Med12 function in the adult nervous system.
Asunto(s)
Astrocitos/fisiología , Pérdida Auditiva Sensorineural/etiología , Complejo Mediador/deficiencia , Estría Vascular/patología , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Moléculas de Adhesión Celular/metabolismo , Condicionamiento Clásico/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico , Miedo , Femenino , Reacción Cataléptica de Congelación , Técnicas de Inactivación de Genes , Pérdida Auditiva Sensorineural/patología , Pérdida Auditiva Sensorineural/fisiopatología , Masculino , Complejo Mediador/fisiología , Ratones , Especificidad de Órganos , Emisiones Otoacústicas Espontáneas , Distribución Aleatoria , Reflejo de SobresaltoRESUMEN
Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.
Asunto(s)
Vías Auditivas/fisiopatología , Disfunción Cognitiva/metabolismo , Eritrocitos/metabolismo , Hipoxia/metabolismo , Receptor de Adenosina A2B/metabolismo , 2,3-Difosfoglicerato/metabolismo , Envejecimiento/patología , Animales , Bisfosfoglicerato Mutasa/genética , Bisfosfoglicerato Mutasa/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Cóclea/fisiopatología , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Activación Enzimática , Eliminación de Gen , Glucólisis , Hipoxia/complicaciones , Hipoxia/genética , Hipoxia/fisiopatología , Inflamación/complicaciones , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/patología , Receptor de Adenosina A2B/deficienciaRESUMEN
Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown. We report that Ada-/- mice, phenocopying ADA-deficient humans, displayed SNHL. Ada-/- mice cochlea with elevated adenosine caused substantial nerve fiber demyelination and mild hair cell loss. ADA enzyme therapy in these mice normalized cochlear adenosine levels, attenuated SNHL, and prevented demyelination. Additionally, ADA enzyme therapy rescued SNHL by restoring nerve fiber structure in Ada-/- mice post two-week drug withdrawal. Moreover, elevated cochlear adenosine in untreated mice was associated with enhanced Adora2b gene expression. Preclinically, ADORA2B-specific antagonist treatment in Ada-/- mice significantly improved HL, nerve fiber density, and myelin compaction. We also provided genetic evidence that ADORA2B is detrimental for age-related SNHL by impairing cochlear myelination in WT aged mice. Overall, understanding purinergic molecular signaling in SNHL in Ada-/- mice allows us to further discover that ADORA2B is also a pathogenic factor underlying aged-related SNHL by impairing cochlear myelination and lowering cochlear adenosine levels or blocking ADORA2B signaling are effective therapies for SNHL.
Asunto(s)
Pérdida Auditiva Sensorineural/metabolismo , Receptor de Adenosina A2B/metabolismo , Factores de Virulencia/metabolismo , Adenosina/metabolismo , Animales , Cóclea/metabolismo , Expresión Génica/fisiología , Células Ciliadas Auditivas/metabolismo , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Fibras Nerviosas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the "omics" family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other "ome" and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands' cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.
Asunto(s)
Anemia de Células Falciformes , Eritrocitos Anormales , Metabolómica , Proteómica , Transducción de Señal , Terapias en Investigación , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Anemia de Células Falciformes/terapia , Animales , Eritrocitos Anormales/metabolismo , Eritrocitos Anormales/patología , Hemoglobina Falciforme/metabolismo , Humanos , RatonesRESUMEN
Erythrocytes are vital to human adaptation under hypoxic conditions because of their abundance in number and irreplaceable function of delivering oxygen (O2). However, although multiple large-scale altitude studies investigating the overall coordination of the human body for hypoxia adaptation have been conducted, detailed research with a focus on erythrocytes was missing due to lack of proper techniques. The recently maturing metabolomics profiling technology appears to be the answer to this limitation. Metabolomics profiling provides unbiased high-throughput screening data that reveal the overall metabolic status of erythrocytes. Recent studies have exploited this new technology and provided novel insight into erythrocyte physiology and pathology. In particular, a series of studies focusing on erythrocyte purinergic signaling have reported that adenosine signaling, coupled with 5' AMP-activated protein kinase (AMPK) and the production of erythrocyte-enriched bioactive signaling lipid sphingosine 1-phosphate, regulate erythrocyte glucose metabolism for more O2 delivery. Moreover, an adenosine-dependent "erythrocyte hypoxic memory" was discovered that provides an explanation for fast acclimation upon re-ascent. These findings not only shed new light on our understanding of erythrocyte function and hypoxia adaptation, but also offer a myriad of novel therapeutic possibilities to counteract various hypoxic conditions.
Asunto(s)
Adaptación Fisiológica/fisiología , Adenosina/metabolismo , Eritrocitos/metabolismo , Hipoxia/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Aclimatación , Animales , Humanos , Oxígeno/metabolismoRESUMEN
Lineage development is a stepwise process, governed by stage-specific regulatory factors and associated markers. Astrocytes are one of the principle cell types in the CNS and the stages associated with their development remain very poorly defined. To identify these stages, we performed gene-expression profiling on astrocyte precursor populations in the spinal cord, identifying distinct patterns of gene induction during their development that are strongly correlated with human astrocytes. Validation studies identified a new cohort of astrocyte-associated genes during development and demonstrated their expression in reactive astrocytes in human white matter injury (WMI). Functional studies on one of these genes revealed that mice lacking Asef exhibited impaired astrocyte differentiation during development and repair after WMI, coupled with compromised blood-brain barrier integrity in the adult CNS. These studies have identified distinct stages of astrocyte lineage development associated with human WMI and, together with our functional analysis of Asef, highlight the parallels between astrocyte development and their reactive counterparts associated with injury. SIGNIFICANCE STATEMENT: Astrocytes play a central role in CNS function and associated diseases. Yet the mechanisms that control their development remain poorly defined. Using the developing mouse spinal cord as a model system, we identify molecular changes that occur in developing astrocytes. These molecular signatures are strongly correlated with human astrocyte expression profiles and validation in mouse spinal cord identifies a host of new genes associated with the astrocyte lineage. These genes are present in reactive astrocytes in human white matter injury, and functional studies reveal that one of these genes, Asef, contributes to reactive astrocyte responses after injury. These studies identify distinct stages of astrocyte lineage development and highlight the parallels between astrocyte development and their reactive counterparts associated with injury.