RESUMEN
Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). The severe forms of GD are associated with neurodegeneration with either rapid (Type 2) or slow progression (Type 3). Although the neurodegenerative process in GD has been linked to lysosomal dysfunction, the mechanisms involved are largely unknown. To identify the lysosomal alterations in GD neurons and uncover the mechanisms involved, we used induced pluripotent stem cells (iPSCs) derived from patients with GD. In GD iPSC-derived neuronal cells (iPSC-NCs), GBA1 mutations caused widespread lysosomal depletion, and a block in autophagic flux due to defective lysosomal clearance of autophagosomes. Autophagy induction by rapamycin treatment in GD iPSC-NCs led to cell death. Further analysis showed that in GD iPSC-NCs, expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes, and lysosomal gene expression, were significantly downregulated. There was also reduced stability of the TFEB protein and altered lysosomal protein biosynthesis. Treatment of mutant iPSC-NCs with recombinant GCase (rGCase) reverted the lysosomal depletion and autophagy block. The effect of rGCase on restoring lysosomal numbers in mutant cells was enhanced in the presence of overexpressed TFEB, but TFEB overexpression alone did not reverse the lysosomal depletion phenotype. Our results suggest that GBA1 mutations interfere with TFEB-mediated lysosomal biogenesis, and that the action of GCase in maintaining a functioning pool of lysosomes is exerted in part through TFEB. The lysosomal alterations described here are likely to be a major determinant in GBA1-associated neurodegeneration.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Enfermedad de Gaucher/fisiopatología , Glucosilceramidasa/genética , Lisosomas/fisiología , Neuronas/fisiología , Biogénesis de Organelos , Diferenciación Celular , Enfermedad de Gaucher/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Lisosomas/metabolismo , Mutación , Neuronas/metabolismoRESUMEN
Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-glucocerebrosidase (GCase; GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the sources of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and whether their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human induced pluripotent stem cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of tumor necrosis factor α, IL-6, and IL-1ß than control cells, and this phenotype was exacerbated by treatment with lipopolysaccharide. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of red blood cell remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant GCase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery.
Asunto(s)
Citocinas/biosíntesis , Enfermedad de Gaucher/metabolismo , Enfermedad de Gaucher/patología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Femenino , Enfermedad de Gaucher/genética , Humanos , Células Madre Pluripotentes Inducidas/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.
Asunto(s)
Enfermedad de Gaucher/patología , Células Madre Pluripotentes/citología , Diferenciación Celular , Linaje de la Célula , Humanos , Activación de Macrófagos , Modelos BiológicosRESUMEN
Mutations in the GBA1 gene are the single most frequent genetic risk factor for Parkinson's disease (PD). Neurodegenerative changes in GBA1-associated PD have been linked to the defective lysosomal clearance of autophagic substrates and aggregate-prone proteins. To elucidate novel mechanisms contributing to proteinopathy in PD, we investigated the effect of GBA1 mutations on the transcription factor EB (TFEB), the master regulator of the autophagy-lysosomal pathway (ALP). Using PD patients' induced-pluripotent stem cells (iPSCs), we examined TFEB activity and regulation of the ALP in dopaminergic neuronal cultures generated from iPSC lines harboring heterozygous GBA1 mutations and the CRISPR/Cas9-corrected isogenic controls. Our data showed a significant decrease in TFEB transcriptional activity and attenuated expression of many genes in the CLEAR network in GBA1 mutant neurons, but not in the isogenic gene-corrected cells. In PD neurons, we also detected increased activity of the mammalian target of rapamycin complex1 (mTORC1), the main upstream negative regulator of TFEB. Increased mTORC1 activity resulted in excess TFEB phosphorylation and decreased nuclear translocation. Pharmacological mTOR inhibition restored TFEB activity, decreased ER stress and reduced α-synuclein accumulation, indicating improvement of neuronal protiostasis. Moreover, treatment with the lipid substrate reducing compound Genz-123346, decreased mTORC1 activity and increased TFEB expression in the mutant neurons, suggesting that mTORC1-TFEB alterations are linked to the lipid substrate accumulation. Our study unveils a new mechanism contributing to PD susceptibility by GBA1 mutations in which deregulation of the mTORC1-TFEB axis mediates ALP dysfunction and subsequent proteinopathy. It also indicates that pharmacological restoration of TFEB activity could be a promising therapeutic approach in GBA1-associated neurodegeneration.
RESUMEN
WT1 is a zinc finger transcription factor expressed at high levels in many types of solid tumors, and high WT1 expression is an adverse prognostic factor. How WT1 contributes to tumor growth and influences prognosis remains unclear. We investigated the hypothesis that WT1 up-regulates VEGF in solid tumors, augmenting the response to hypoxia. We found a correlation between levels of WT1 expression and VEGF expression in Ewing sarcoma cell lines. Transfecting WT1-null SK-ES-1 cells with WT1 up-regulated VEGF mRNA expression and resulted in increased angiogenic activity in vitro. Conversely, diminishing WT1 expression in WT1-positive cell lines using WT1-specific shRNA down-regulated VEGF mRNA expression and decreased angiogenic activity in vitro. Transient transfection assays demonstrated that WT1 can regulate the activity of the VEGF promoter, and chromatin immunoprecipitation assays showed that WT1 can bind directly to the VEGF promoter in intact cells. WT1 expression in Ewing sarcoma cells is up-regulated by hypoxia. Importantly, using shRNA to inhibit this up-regulation blunted the hypoxia-mediated increase in VEGF expression. Taken together, these data demonstrate that VEGF is a direct, bona fide WT1 target gene in sarcoma and that WT1 plays a key role in optimizing the response of tumor cells to hypoxia.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Sarcoma de Ewing/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas WT1/metabolismo , Animales , Progresión de la Enfermedad , Células Endoteliales/citología , Genes Supresores de Tumor , Humanos , Hipoxia , Ratones , Células 3T3 NIH , Neovascularización Patológica , Pronóstico , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Venas Umbilicales/citologíaRESUMEN
Exogenous bone marrow-derived cells (BMDCs) are promising therapeutic agents for the treatment of tissue ischemia and traumatic injury. However, until we identify the molecular mechanisms that underlie their actions, there can be no rational basis for the design of therapeutic strategies using BMDCs. The pro-healing effects of BMDCs are apparent very shortly after treatment, which suggests that they may exert their effects by the modulation of acute inflammation. We investigated this hypothesis by taking advantage of the fact that BMDCs from healthy, young, but not obese, diabetic mice stimulate vascular growth. By comparing both in vitro secretion and in vivo local induction of acute phase inflammatory cytokines by these cells, we identified monocyte chemoattractant factor 1 and tumor necrosis factor α as potential mediators of BMDC-induced tissue repair. In vivo analysis of BMDC-treated ischemic limbs and cutaneous wounds revealed that the production of monocyte chemoattractant factor 1 by exogenous and endogenous BMDCs is essential for BMDC-mediated vascular growth and tissue healing, while the inability of BMDCs to produce tumor necrosis factor α appears to play a lesser but still meaningful role. Thus, measurements of the secretion of cytokines by BMDCs may allow us to identify a priori individuals who would or would not be good candidates for BMDC-based therapies.
Asunto(s)
Médula Ósea/metabolismo , Médula Ósea/patología , Quimiocina CCL2/metabolismo , Inflamación/prevención & control , Isquemia/prevención & control , Receptores de Leptina/metabolismo , Cicatrización de Heridas , Animales , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Extremidades , Técnicas para Inmunoenzimas , Inflamación/etiología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Isquemia/etiología , Isquemia/metabolismo , Ratones , Ratones Noqueados , Piel/lesiones , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, the gene that encodes lysosomal ß-glucocerebrosidase (GCase). Mild mutations in GBA1 cause type 1 non-neuronopathic GD, whereas severe mutations cause types 2 and 3 neuronopathic GD (nGD). GCase deficiency results in the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). GlcSph is formed by deacylation of GlcCer by the lysosomal enzyme acid ceramidase. Brains from patients with nGD have high levels of GlcSph, a lipid believed to play an important role in nGD, but the mechanisms involved remain unclear. To identify these mechanisms, we used human induced pluripotent stem cell-derived neurons from nGD patients. We found that elevated levels of GlcSph activate mammalian target of rapamycin (mTOR) complex 1 (mTORC1), interfering with lysosomal biogenesis and autophagy, which were restored by incubation of nGD neurons with mTOR inhibitors. We also found that inhibition of acid ceramidase prevented both, mTOR hyperactivity and lysosomal dysfunction, suggesting that these alterations were caused by GlcSph accumulation in the mutant neurons. To directly determine whether GlcSph can cause mTOR hyperactivation, we incubated wild-type neurons with exogenous GlcSph. Remarkably, GlcSph treatment recapitulated the mTOR hyperactivation and lysosomal abnormalities in mutant neurons, which were prevented by coincubation of GlcSph with mTOR inhibitors. We conclude that elevated GlcSph activates an mTORC1-dependent pathogenic mechanism that is responsible for the lysosomal abnormalities of nGD neurons. We also identify acid ceramidase as essential to the pathogenesis of nGD, providing a new therapeutic target for treating GBA1-associated neurodegeneration.
Asunto(s)
Enfermedad de Gaucher , Células Madre Pluripotentes Inducidas , Diana Mecanicista del Complejo 1 de la Rapamicina , Neuronas , Psicosina/análogos & derivados , Ceramidasa Ácida/antagonistas & inhibidores , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Lisosomas , Inhibidores mTOR , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/citología , Psicosina/sangreRESUMEN
Recent studies indicate a causative relationship between defects in autophagy and dopaminergic neuron degeneration in Parkinson disease (PD). However, it is not fully understood how autophagy is regulated in the context of PD. Here we identify USP24 (ubiquitin specific peptidase 24), a gene located in the PARK10 (Parkinson disease 10 [susceptibility]) locus associated with late onset PD, as a novel negative regulator of autophagy. Our data indicate that USP24 regulates autophagy by affecting ubiquitination and stability of the ULK1 protein. Knockdown of USP24 in cell lines and in human induced-pluripotent stem cells (iPSC) differentiated into dopaminergic neurons resulted in elevated ULK1 protein levels and increased autophagy flux in a manner independent of MTORC1 but dependent on the class III phosphatidylinositol 3-kinase (PtdIns3K) activity. Surprisingly, USP24 knockdown also improved neurite extension and/or maintenance in aged iPSC-derived dopaminergic neurons. Furthermore, we observed elevated levels of USP24 in the substantia nigra of a subpopulation of idiopathic PD patients, suggesting that USP24 may negatively regulate autophagy in PD.Abbreviations: Bafilomycin/BafA: bafilomycin A1; DUB: deubiquitinating enzyme; iPSC: induced pluripotent stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; nt: non-targeting; PD: Parkinson disease; p-ATG13: phospho-ATG13; PtdIns3P: phosphatidylinositol 3-phosphate; RPS6: ribosomal protein S6; SNPs: single nucleotide polymorphisms; TH: tyrosine hydroxylase; USP24: ubiquitin specific peptidase 24.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedad de Parkinson/genética , Ubiquitina Tiolesterasa/genética , Autofagia/genética , Autofagia/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad de Parkinson/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.
Asunto(s)
Enfermedad de Gaucher/patología , Células Madre Pluripotentes Inducidas/patología , Lisosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/metabolismo , Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biomarcadores/metabolismo , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lípidos/química , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Naftiridinas/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Estabilidad Proteica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: Peripheral blood contains primitive (stem cell-like) and monocytic-like endothelial cell progenitors. Diabetes apparently converts these primitive progenitors, from a pro-angiogenic to anti-angiogenic phenotype. Monocytic progenitors seem to be less affected by diabetes, but potential pro-angiogenic activities of freshly isolated monocytic progenitors remain unexplored. We compared the ability of primitive and monocytic endothelial cell progenitors to stimulate vascular growth and healing in diabetes and investigated potential molecular mechanisms through which the cells mediate their in vivo effects. METHODS AND RESULTS: Human CD34+ primitive progenitors and CD14+ monocytic progenitors were injected locally into the ischemic limbs of diabetic mice. CD14+ cell therapy improved healing and vessel growth, although not as rapidly or effectively as CD34+ cell treatment. Western blot analysis revealed that cell therapy modulated expression of molecules in the VEGF, MCP-1, and angiopoietin pathways. CONCLUSIONS: Injection of freshly isolated circulating CD14+ cells improves healing and vascular growth indicating their potential for use in acute clinical settings. Importantly, CD14+ cells could provide a therapeutic option for people with diabetes, the function of whose CD34+ cells may be compromised. At least some progenitor-induced healing probably is mediated through increased sensitivity to VEGF and increases in MCP-1, and possibly modulation of angiopoietins.
Asunto(s)
Diferenciación Celular , Diabetes Mellitus Experimental/fisiopatología , Endotelio Vascular/fisiología , Células Madre Hematopoyéticas/fisiología , Monocitos/fisiología , Neovascularización Fisiológica , Animales , Antígenos CD34 , Células Cultivadas , Diabetes Mellitus Experimental/patología , Endotelio Vascular/patología , Extremidades/irrigación sanguínea , Células Madre Hematopoyéticas/patología , Humanos , Isquemia/terapia , Receptores de Lipopolisacáridos , Masculino , Ratones , Ratones Desnudos , Monocitos/patología , Trasplante de Células Madre , Cicatrización de HeridasRESUMEN
Gaucher's disease (GD) is an autosomal recessive disorder caused by mutations in the GBA1 gene, which encodes acid ß-glucocerebrosidase (GCase). Severe GBA1 mutations cause neuropathology that manifests soon after birth, suggesting that GCase deficiency interferes with neuronal development. We found that neuronopathic GD induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs) exhibit developmental defects due to downregulation of canonical Wnt/ß-catenin signaling and that GD iPSCs' ability to differentiate to dopaminergic (DA) neurons was strikingly reduced due to early loss of DA progenitors. Incubation of the mutant cells with the Wnt activator CHIR99021 (CHIR) or with recombinant GCase restored Wnt/ß-catenin signaling and rescued DA differentiation. We also found that GD NPCs exhibit lysosomal dysfunction, which may be involved in Wnt downregulation by mutant GCase. We conclude that neuronopathic mutations in GCase lead to neurodevelopmental abnormalities due to a critical requirement of this enzyme for canonical Wnt/ß-catenin signaling at early stages of neurogenesis.
Asunto(s)
Diferenciación Celular/genética , Neuronas Dopaminérgicas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Neurogénesis/genética , Neuronas Dopaminérgicas/patología , Enfermedad de Gaucher/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lisosomas/genética , Lisosomas/patología , Mutación , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/patología , Piridinas/administración & dosificación , Pirimidinas/administración & dosificación , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Bone marrow stem cells participate in tissue repair processes and may have roles in skin wound repair. Diabetes is characterized by delayed and poor wound healing, and type 1 diabetes seems to lead to stem cell dysfunction. Hence, stem cell dysfunction could contribute to poor healing, and stem cell-based therapies may be efficacious in diabetic wounds. We investigated the potential of exogenous stem cells to promote skin healing and possible effects of type 2 diabetes on stem cell function. Mouse bone marrow cells from nondiabetic and diabetic mice were enriched for putative stem cells and injected under skin wounds of nondiabetic or type 2 diabetic Leprdb mice. Using histology and morphometry, vascularization and healing in treated and untreated mice were analyzed. We anticipated a correlation between improved wound healing and vascularization, because therapies that increase tissue vascularization tend to enhance wound healing. Our data indicate that exogenous nondiabetic bone marrow-derived cells increase vascularization and improve wound healing in Leprdb mice but have little effect on nondiabetic controls. In contrast, Leprdb-derived marrow cells inhibit vascularization but promote wound healing in Leprdb mice. Thus, adult stem cell function may be impaired by type 2 diabetes; the ability to promote vascularization and wound healing are distinct functions of bone marrow cells; and neovascularization and wound healing may not be tightly coupled. Additionally, we observed little incorporation of injected cells into wound structures, suggesting that improved healing is mediated through mechanisms other than direct differentiation and incorporation of the cells.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Trasplante de Células Madre Hematopoyéticas , Neovascularización Fisiológica , Receptores de Superficie Celular/genética , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas , Animales , Células Madre Hematopoyéticas/fisiología , Inflamación/etiología , Inyecciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores de Leptina , Piel/irrigación sanguínea , Piel/lesiones , Piel/patologíaRESUMEN
Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.
Asunto(s)
Glucosilceramidasa/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Células Cultivadas , Diseño de Fármacos , Glucosilceramidasa/genética , Células HEK293 , Humanos , Neuronas/citología , Psicosina/análogos & derivados , Psicosina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Gaucher disease (GD) is the most common lysosomal storage disease resulting from mutations in the lysosomal enzyme glucocerebrosidase (GCase). The hematopoietic abnormalities in GD include the presence of characteristic Gaucher macrophages that infiltrate patient tissues and cytopenias. At present, it is not clear whether these cytopenias are secondary to the pathological activity of Gaucher cells or a direct effect of GCase deficiency on hematopoietic development. To address this question, we differentiated induced pluripotent stem cells (iPSCs) derived from patients with types 1, 2, and 3 GD to CD34(+)/CD45(+)/CD43(+)/CD143(+) hematopoietic progenitor cells (HPCs) and examined their developmental potential. The formation of GD-HPCs was unaffected. However, these progenitors demonstrated a skewed lineage commitment, with increased myeloid differentiation and decreased erythroid differentiation and maturation. Interestingly, myeloid colony-formation assays revealed that GD-HPCs, but not control-HPCs, gave rise to adherent, macrophage-like cells, another indication of abnormal myelopoiesis. The extent of these hematologic abnormalities correlated with the severity of the GCase mutations. All the phenotypic abnormalities of GD-HPCs observed were reversed by incubation with recombinant GCase, indicating that these developmental defects were caused by the mutated GCase. Our results show that GCase deficiency directly impairs hematopoietic development. Additionally, our results suggest that aberrant myelopoiesis might contribute to the pathological properties of Gaucher macrophages, which are central to GD manifestations. The hematopoietic developmental defects we observed reflect hematologic abnormalities in patients with GD, demonstrating the utility of GD-iPSCs for modeling this disease.
Asunto(s)
Enfermedad de Gaucher/terapia , Hematopoyesis , Células Madre Pluripotentes Inducidas/trasplante , Mielopoyesis/fisiología , Diferenciación Celular , Linaje de la Célula , Enfermedad de Gaucher/patología , Células Madre Hematopoyéticas/patología , Humanos , Macrófagos/patologíaRESUMEN
After decades of speculation, proof of embryonic hemangioblasts finally emerged a few years ago. Surprisingly, at about the same time, evidence for adult hemangioblasts began to appear, and recent single-cell bone marrow transplants have confirmed their existence. Embryonic and adult hemangioblasts appear to share antigenic determinants, including CD34, ACC133, and VEGFR2, although their phenotype may be plastic. They also respond to similar factors, prominent among them vascular endothelial growth factor (VEGF). In the adult, hemangioblasts reside principally in the bone marrow, although they may subsequently leave that niche to reside in nonhematopoietic tissues. A number of studies indicate that these cells or their progeny may be a significant source of endothelial cells in adult pathologic and nonpathologic vascularization, and may participate in vascular repair. In addition to hemangioblasts, a more differentiated source of endothelial cell progenitors may be present in the blood, namely, monocytes or monocytic-like cells. The relative importance of the two cell types in vivo is not clear, though endothelial cells derived from the two sources may not be identical, and hemangioblasts seem to provide a stimulus for differentiation of the monocytes. Treatment with exogenous bone marrow-derived cells can promote neovascularization, accelerate restoration of blood flow to ischemic tissues, and improve cardiac function after infarct. Hence, there is great hope that either alone, in combination with angiogenic factors, or as gene therapy vectors, we can harness these cells to treat ischemic and vascular diseases in the relatively near future.
Asunto(s)
Endotelio Vascular/citología , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Animales , HumanosRESUMEN
Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.
Asunto(s)
Cordoma/tratamiento farmacológico , Cordoma/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , FN-kappa B/metabolismo , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cordoma/genética , Perfilación de la Expresión Génica , Genotipo , Humanos , Inmunohistoquímica , Ratones , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Trasplante HeterólogoRESUMEN
OBJECT: Chordoma is a malignant bone neoplasm hypothesized to arise from notochordal remnants along the length of the neuraxis. Recent genomic investigation of chordomas has identified T (Brachyury) gene duplication as a major susceptibility mutation in familial chordomas. Brachyury plays a vital role during embryonic development of the notochord and has recently been shown to regulate epithelial-to-mesenchymal transition in epithelial-derived cancers. However, current understanding of the role of this transcription factor in chordoma is limited due to the lack of availability of a fully characterized chordoma cell line expressing Brachyury. Thus, the objective of this study was to establish the first fully characterized primary chordoma cell line expressing gain of the T gene locus that readily recapitulates the original parental tumor phenotype in vitro and in vivo. METHODS: Using an intraoperatively obtained tumor sample from a 61-year-old woman with primary sacral chordoma, a chordoma cell line (JHC7, or Johns Hopkins Chordoma Line 7) was established. Molecular characterization of the primary tumor and cell line was conducted using standard immunostaining and Western blotting. Chromosomal aberrations and genomic amplification of the T gene in this cell line were determined. Using this cell line, a xenograft model was established and the histopathological analysis of the tumor was performed. Silencing of Brachyury and changes in gene expression were assessed. RESULTS: The authors report, for the first time, the successful establishment of a chordoma cell line (JHC7) from a patient with pathologically confirmed sacral chordoma. This cell line readily forms tumors in immunodeficient mice that recapitulate the parental tumor phenotype with conserved histological features consistent with the parental tumor. Furthermore, it is demonstrated for the first time that silencing of Brachyury using short hairpin RNA renders the morphology of chordoma cells to a more differentiated-like state and leads to complete growth arrest and senescence with an inability to be passaged serially in vitro. CONCLUSIONS: This report represents the first xenograft model of a sacral chordoma line described in the literature and the first cell line established with stable Brachyury expression. The authors propose that Brachyury is an attractive therapeutic target in chordoma and that JHC7 will serve as a clinically relevant model for the study of this disease.
Asunto(s)
Neoplasias Óseas/patología , Línea Celular Tumoral/citología , Cordoma/patología , Proteínas Fetales/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/cirugía , Línea Celular Tumoral/metabolismo , Supervivencia Celular , Cordoma/metabolismo , Cordoma/cirugía , Modelos Animales de Enfermedad , Femenino , Proteínas Fetales/genética , Expresión Génica , Humanos , Ratones , Persona de Mediana Edad , Proteínas de Dominio T Box/genética , Trasplante HeterólogoRESUMEN
BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Sarcoma de Ewing/patología , Animales , Antineoplásicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/metabolismo , Sarcoma Experimental/tratamiento farmacológico , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Trasplante Heterólogo , Verapamilo/farmacologíaAsunto(s)
Antígenos CD34/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Endotelio Vascular/embriología , Receptores de Lipopolisacáridos/metabolismo , Neovascularización Fisiológica/inmunología , Células Madre/metabolismo , Animales , Antígenos CD34/inmunología , Factores de Crecimiento Endotelial/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Receptores de Lipopolisacáridos/inmunología , Linfocinas/metabolismo , Linfocinas/farmacología , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Células Madre/citología , Células Madre/inmunología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Two classes of adult bone marrow-derived endothelial cell (EC) progenitors have been described, primitive hematopoietic stem cell-related cells and monocytic cells. Both differentiate into ECs and promote vascular growth in vivo but have distinct characteristics. Despite the association of obesity and type 2 diabetes with cardiovascular disease, their effects on primitive EC progenitors (prECPs) have not been examined, and the limited data on monocytic EC progenitors are conflicting. We investigated functional parameters of primitive and monocytic EC progenitors from obese diabetic (Lepr(db)) mice. The viability, proliferation, and differentiation of EC progenitors were unaffected in Lepr(db) cell cultures under basal condition. However, Lepr(db)-derived prECPs, but not monocytic EC progenitors, were less able to cope with hypoxia and oxidative stress, conditions likely present when EC progenitors are most needed. Intrinsic prECP dysfunction was also apparent in vivo. Whereas injection of nondiabetic prECPs promoted vascularization of skin wounds, Lepr(db)-derived progenitors inhibited it in nondiabetic mice. Additionally, although treatment with Lepr(db)-derived prECPs did not significantly reduce blood flow restoration to ischemic limbs, it resulted in increased tissue necrosis and autoamputation. Thus, type 2 diabetes coupled with obesity seems to induce intrinsic EC progenitor dysfunction that is exacerbated by stress. prECPs are more affected than monocytic progenitors, exhibiting a reduced ability to survive or proliferate. The proangiogenic phenotype of prECPs also seems to convert to an antiangiogenic phenotype in obese diabetic mice. These data suggest that therapies involving prECPs or stem-like cells in diabetic patients may be inadvisable at this time.