RESUMEN
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Asunto(s)
Barrera Hematoencefálica , Oligonucleótidos Antisentido , ARN Largo no Codificante , Receptores de Transferrina , Animales , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/administración & dosificación , Barrera Hematoencefálica/metabolismo , Receptores de Transferrina/metabolismo , Humanos , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Ratones , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamiento del Gen , Distribución TisularRESUMEN
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Asunto(s)
Barrera Hematoencefálica , Iduronato Sulfatasa , Animales , Encéfalo , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático , Lisosomas , RatonesRESUMEN
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Barrera Hematoencefálica , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Haplorrinos/metabolismo , Fragmentos Fc de Inmunoglobulinas , Ratones , Receptores de Transferrina/metabolismoRESUMEN
The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFß. On-target activity was demonstrated by the Runt domain inhibitors' ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.
Asunto(s)
Antineoplásicos/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/genética , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Estructura Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Translocación Genética , Pez CebraRESUMEN
Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease.
Asunto(s)
Neoplasias Hematológicas/patología , Hematopoyesis , Pez Cebra/fisiología , Animales , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/embriología , Pez Cebra/embriologíaRESUMEN
Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRß-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRß signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRß regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRß and IL-6/IL-6R that acts to control embryonic HSPC production.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia , Interleucina-6/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Proliferación Celular , Expresión Génica , Células Madre Hematopoyéticas/citología , Inmunohistoquímica , Estabilidad Proteica , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Pez CebraRESUMEN
Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8(IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Células Madre Hematopoyéticas/metabolismo , Interleucina-8/genética , Receptores de Calcitriol/genética , Vitamina D/genética , Proteínas de Pez Cebra/genética , Animales , Disponibilidad Biológica , Señalización del Calcio/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Humanos , Interleucina-8/metabolismo , Embarazo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Vitamina D/metabolismo , Deficiencia de Vitamina D/genética , Deficiencia de Vitamina D/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers.
Asunto(s)
Sistema Nervioso Central/metabolismo , Células Madre Embrionarias/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Animales , Recuento de Células , Hipoxia de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Fluoxetina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/metabolismo , Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Triptófano Hidroxilasa/metabolismo , Pez Cebra/embriologíaRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.
Asunto(s)
Colecalciferol/farmacología , Proteínas Hedgehog/metabolismo , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Vitaminas/farmacología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Colecalciferol/metabolismo , Colestanotriol 26-Monooxigenasa/genética , Colestanotriol 26-Monooxigenasa/metabolismo , Eliminación de Gen , Células Madre Hematopoyéticas/citología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Smoothened , Vitaminas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.
Asunto(s)
Dinoprostona/metabolismo , Células Madre Hematopoyéticas/metabolismo , Selectina-P/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Células Madre Hematopoyéticas/citología , Transducción de Señal/fisiologíaRESUMEN
Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-Ï activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Transducción de Señal , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Proliferación Celular/genética , Células Cultivadas , Citocinas/inmunología , Embrión de Mamíferos , Embrión no Mamífero , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/genética , Inflamación/inmunología , Interferones/genética , Interferones/metabolismo , Ratones , Pez Cebra/embriologíaRESUMEN
Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.
Asunto(s)
Antagonistas de Estrógenos/farmacología , Hemangioblastos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Compuestos de Bencidrilo/farmacología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Efrina-B2/antagonistas & inhibidores , Estradiol/análogos & derivados , Estradiol/farmacología , Estrógenos/farmacología , Etinilestradiol/farmacología , Fulvestrant , Genisteína/farmacología , Respuesta al Choque Térmico , Morfolinos/genética , Fenoles/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Estradiol/genética , Receptores Notch/biosíntesis , Transducción de Señal , Proteína 1 de la Leucemia Linfocítica T Aguda , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed.
Asunto(s)
Quimiotaxis , Enfermedad de Huntington/genética , Microglía/fisiología , Células Mieloides/fisiología , Proteínas del Tejido Nervioso/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/fisiología , Animales , Extensiones de la Superficie Celular/metabolismo , Células Cultivadas , Complemento C5a/fisiología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/patología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/citología , Monocitos/fisiología , Mutación , Células Mieloides/citología , Proteínas del Tejido Nervioso/metabolismo , Peritoneo/patología , Tioglicolatos/farmacología , Imagen de Lapso de TiempoRESUMEN
Huntington's disease (HD) is caused by an expanded polyglutamine tract in the protein huntingtin (htt). Although HD has historically been viewed as a brain-specific disease, htt is expressed ubiquitously, and recent studies indicate that mutant htt might cause changes to the immune system that could contribute to pathogenesis. Monocytes from HD patients and mouse models are hyperactive in response to stimulation, and increased levels of inflammatory cytokines and chemokines are found in pre-manifest patients that correlate with pathogenesis. In this study, wild-type (WT) bone marrow cells were transplanted into two lethally irradiated transgenic mouse models of HD that ubiquitously express full-length htt (YAC128 and BACHD mice). Bone marrow transplantation partially attenuated hypokinetic and motor deficits in HD mice. Increased levels of synapses in the cortex were found in HD mice that received bone marrow transplants. Importantly, serum levels of interleukin-6, interleukin-10, CXC chemokine ligand 1, and interferon-γ were significantly higher in HD than WT mice but were normalized in mice that received a bone marrow transplant. These results suggest that immune cell dysfunction might be an important modifier of pathogenesis in HD.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/terapia , Células de la Médula Ósea/inmunología , Trasplante de Médula Ósea/métodos , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/terapia , Terapia de Inmunosupresión/métodos , Animales , Enfermedades Autoinmunes del Sistema Nervioso/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/toxicidad , Proteínas Nucleares/metabolismo , Proteínas Nucleares/toxicidad , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Secuencia de Bases , Eliminación de Gen , Genómica , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta/genética , ARN Ribosómico/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/citología , Transgenes/genéticaRESUMEN
Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Sarcoma de Células Claras/enzimología , Sarcoma de Células Claras/patología , Benzamidas/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Depsipéptidos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sarcoma de Células Claras/genética , Esferoides Celulares/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Translocación Genética/efectos de los fármacosRESUMEN
The kynurenine pathway of tryptophan degradation is hypothesized to play an important role in Huntington disease, a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin. Neurotoxic metabolites of the kynurenine pathway, generated in microglia and macrophages, are present at increased levels in the brains of patients and mouse models during early stages of disease, but the mechanism by which kynurenine pathway up-regulation occurs in Huntington disease is unknown. Here we report that expression of a mutant huntingtin fragment was sufficient to induce transcription of the kynurenine pathway in yeast and that this induction was abrogated by impairing the activity of the histone deacetylase Rpd3. Moreover, numerous genetic suppressors of mutant huntingtin toxicity that are functionally unrelated converged unexpectedly on the kynurenine pathway, supporting a critical role for the kynurenine pathway in mediating mutant huntingtin toxicity in yeast. Histone deacetylase-dependent regulation of the kynurenine pathway was also observed in a mouse model of Huntington disease, in which treatment with a neuroprotective histone deacetylase inhibitor blocked activation of the kynurenine pathway in microglia expressing a mutant huntingtin fragment in vitro and in vivo. These findings suggest that a mutant huntingtin fragment can perturb transcriptional programs in microglia, and thus implicate these cells as potential modulators of neurodegeneration in Huntington disease that are worthy of further investigation.
Asunto(s)
Histona Desacetilasas/metabolismo , Enfermedad de Huntington/metabolismo , Quinurenina/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Mutación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Nucleares/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Expresión Génica , Histona Desacetilasas/genética , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Quinurenina/genética , Macrófagos/patología , Ratones , Microglía/patología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genéticaRESUMEN
Synovial sarcoma is a soft tissue malignancy with a poor prognosis; many patients will die from this disease within 10 years of diagnosis, despite treatment. Gene expression profiling and immunohistochemistry studies have identified oncogenes that are highly expressed in synovial sarcoma. Included in this group are receptor tyrosine kinases such as epidermal growth factor receptor, insulin-like growth factor receptor 1, fibroblast growth factor receptor 3, KIT, and HER2. Inhibitors of these growth-promoting receptors are likely to inhibit proliferation of synovial sarcoma; however, the effect of receptor tyrosine kinase inhibitors on synovial sarcoma is largely unknown. We assessed the ability of the following receptor tyrosine kinase inhibitors to halt proliferation and induce apoptosis in synovial sarcoma monolayer and three dimensional spheroid in vitro models: gefitinib (Iressa), NVP-AEW541, imatinib mesylate (Gleevec), SU5402, PRO-001, trastuzumab (Herceptin), and 17-allylamino-17-demethoxygeldanamycin (17-AAG). Gefitinib, NVP-AEW541, and imatinib inhibited proliferation only at relatively high concentrations, which are not clinically applicable. 17-AAG, which destabilizes multiple receptor tyrosine kinases and other oncoproteins through heat shock protein 90 inhibition, prevented proliferation and induced apoptosis in synovial sarcoma monolayer models at concentrations achievable in human serum. 17-AAG treatment was also associated with receptor tyrosine kinase degradation and induction of apoptosis in synovial sarcoma spheroid models. 17-AAG was more effective than doxorubicin, particularly in the spheroid models. Here we provide in vitro evidence that 17-AAG, a clinically applicable drug with known pharmacology and limited toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis, and thus has potential as a systemic therapy for this disease.