RESUMO
O-GlcNAcylation is thought to play a role in the development of tau pathology in Alzheimer's disease because of its ability to modulate tau's aggregation propensity. O-GlcNAcylation is regulated by 2 enzymes: O-GlcNAc transferase and O-GlcNAcase (OGA). Development of a PET tracer would therefore be an essential tool for developing therapeutic small-molecule inhibitors of OGA, enabling clinical testing of target engagement and dose selection. Methods: A collection of small-molecule compounds was screened for inhibitory activity and high-affinity binding to OGA, as well as favorable PET tracer attributes (multidrug resistance protein 1 efflux, central nervous system PET multiparameter optimization, etc.). Two lead compounds with high affinity and selectivity for OGA were selected for further profiling, including OGA binding to tissue homogenate using a radioligand competition binding assay. In vivo pharmacokinetics were established using a microdosing approach with unlabeled compounds in rats. In vivo imaging studies were performed in rodents and nonhuman primates (NHPs) with 11C-labeled compounds. Results: Two selected candidates, BIO-735 and BIO-578, displayed promising attributes in vitro. After radiolabeling with tritium, [3H]BIO-735 and [3H]BIO-578 binding in rodent brain homogenates demonstrated dissociation constants of 0.6 and 2.3 nM, respectively. Binding was inhibited, concentration-dependently, by homologous compounds and thiamet G, a well-characterized and structurally diverse OGA inhibitor. Imaging studies in rats and NHPs showed both tracers had high uptake in the brain and inhibition of binding to OGA in the presence of a nonradioactive compound. However, only BIO-578 demonstrated reversible binding kinetics within the time frame of a PET study with a 11C-labeled molecule to enable quantification using kinetic modeling. Specificity of tracer uptake was confirmed with a 10 mg/kg blocking dose of thiamet G. Conclusion: We describe the development and testing of 2 11C PET tracers targeting the protein OGA. The lead compound BIO-578 demonstrated high affinity and selectivity for OGA in rodent and human postmortem brain tissue, leading to its further testing in NHPs. NHP PET imaging studies showed that the tracer had excellent brain kinetics, with full inhibition of specific binding by thiamet G. These results suggest that the tracer [11C]BIO-578 is well suited for further characterization in humans.
Assuntos
Encéfalo , beta-N-Acetil-Hexosaminidases , Humanos , Ratos , Animais , PiranosRESUMO
Diabetic kidney disease and atherosclerotic disease are major causes of morbidity and mortality associated with type 2 diabetes (T2D), and diabetic kidney disease is a major cardiovascular risk factor. The black and tan, brachyury (BTBR) mouse strain with leptin deficiency (Lepob) has emerged as one of the best models of human diabetic kidney disease. However, no T2D mouse model of combined diabetic kidney disease and atherosclerosis exists. Our goal was to generate such a model. To this end, the low-density lipoprotein (LDL) receptor was targeted for degradation via inducible degrader of the LDL receptor (IDOL) overexpression, using liver-targeted adenoassociated virus serotype DJ/8 (AAV-DJ/8) in BTBR wild-type and BTBR Lepob mice. Liver-targeted IDOL-AAV-DJ/8 increased plasma LDL cholesterol compared with the control enhanced green fluorescent protein AAV-DJ/8. IDOL-induced dyslipidemia caused formation of atherosclerotic lesions of an intermediate stage, which contained both macrophages and smooth muscle cells. BTBR Lepob mice exhibited diabetic kidney disease. IDOL-induced dyslipidemia worsened albuminuria and glomerular macrophage accumulation but had no effect on mesangial expansion or podocyte numbers. Thus, by inducing hepatic degradation of the LDL receptor, we generated a T2D model of combined kidney disease and atherosclerosis. This model provides a new tool to study mechanisms, interactions, and treatment strategies of kidney disease and atherosclerosis in T2D.
Assuntos
Aterosclerose/etiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/etiologia , Animais , Aterosclerose/sangue , Aterosclerose/patologia , Colesterol/sangue , Dependovirus/genética , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Nefropatias Diabéticas/sangue , Modelos Animais de Doenças , Dislipidemias/sangue , Dislipidemias/complicações , Vetores Genéticos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores de LDL/biossíntese , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Neuronal ApoE receptors are linked to learning and memory, but the pathways governing their abundance, and the mechanisms by which they affect the function of neural circuits are incompletely understood. Here we demonstrate that the E3 ubiquitin ligase IDOL determines synaptic ApoER2 protein levels in response to neuronal activation and regulates dendritic spine morphogenesis and plasticity. IDOL-dependent changes in ApoER2 abundance modulate dendritic filopodia initiation and synapse maturation. Loss of IDOL in neurons results in constitutive overexpression of ApoER2 and is associated with impaired activity-dependent structural remodeling of spines and defective LTP in primary neuron cultures and hippocampal slices. IDOL-deficient mice show profound impairment in experience-dependent reorganization of synaptic circuits in the barrel cortex, as well as diminished spatial and associative learning. These results identify control of lipoprotein receptor abundance by IDOL as a post-transcriptional mechanism underlying the structural and functional plasticity of synapses and neural circuits.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Aprendizagem , Plasticidade Neuronal/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Escala de Avaliação Comportamental , Condicionamento Clássico , Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Sinapses/metabolismoRESUMO
Apolipoprotein E (ApoE) is an important modifier of Alzheimer's disease (AD) pathogenesis, and its abundance has been linked to the clearance of ß-amyloid (Aß) in the brain. The pathways that control the clearance of ApoE in the brain are incompletely understood. We report that Idol, an E3 ubiquitin ligase that targets the low-density lipoprotein receptor (LDLR) for degradation, is a critical determinant of brain ApoE metabolism and Aß plaque biogenesis. Previous work has shown that Idol contributes minimally to the regulation of hepatic LDLR expression in mice. By contrast, we demonstrate that Idol is a primary physiological regulator of LDLR protein in the brain, controlling the clearance of both ApoE-containing high-density lipoprotein (HDL) particles and Aß. We studied the consequences of loss of Idol expression in a transgenic mouse model of Aß amyloidosis. Idol deficiency increased brain LDLR, decreased ApoE, decreased soluble and insoluble Aß, reduced amyloid plaque burden, and ameliorated neuroinflammation. These findings identify Idol as a gatekeeper of LDLR-dependent ApoE and Aß clearance in the brain and a potential enzyme target for therapeutic intervention in AD.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Amiloidose/enzimologia , Apolipoproteínas E/metabolismo , Encéfalo/enzimologia , Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/genética , Amiloidose/genética , Amiloidose/patologia , Amiloidose/terapia , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Placa Amiloide , Proteólise , Fatores de Tempo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
The DNA-binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/ß-catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, ß-catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced ß-catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/ß-catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.
Assuntos
Regulação da Expressão Gênica , Homeostase do Telômero , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Via de Sinalização Wnt , Animais , Sítios de Ligação , Feminino , Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Transcriptoma , beta Catenina/metabolismoRESUMO
The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.
Assuntos
Endocitose , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Fosfoproteínas/metabolismo , Receptores de LDL/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Clatrina/metabolismo , Proteínas de Ligação a DNA/genética , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Camundongos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno , Receptores de LDL/genética , Serina Endopeptidases , Fatores de Transcrição/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.
Assuntos
Disceratose Congênita/genética , Disceratose Congênita/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Telômero/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Reprogramação Celular , Fibroblastos , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologia , Telômero/genética , Telômero/metabolismoRESUMO
In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.
Assuntos
Modelos Animais de Doenças , Camundongos , Distrofia Muscular de Duchenne/genética , Células-Tronco/metabolismo , Telômero/metabolismo , Animais , Proliferação de Células , Distrofina/metabolismo , Humanos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/genética , PreconceitoRESUMO
Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.
Assuntos
Cromatina/genética , Transdução de Sinais , Telomerase/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Coristoma/genética , Coristoma/patologia , DNA Helicases/metabolismo , Genes Reporter/genética , Células HeLa , Humanos , Intestino Delgado/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Somitos/anormalidades , Somitos/embriologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Xenopus laevis/embriologia , beta Catenina/genéticaRESUMO
Stem cells age, but the underlying mechanisms remain unclear. In a recent issue of Cell, Inomata and colleagues (2009) show that DNA damage, a prime suspect in stem cell aging, causes graying and loss of melanocyte stem cells by inducing premature differentiation, without inducing apoptosis or senescence.
Assuntos
Diferenciação Celular , Senescência Celular , Nicho de Células-Tronco/citologia , Células-Tronco/citologia , Animais , Dano ao DNA , Cabelo/metabolismo , Óperon Lac , Melaninas/metabolismo , Melanócitos/metabolismo , Camundongos , Nicho de Células-Tronco/metabolismo , Células-Tronco/metabolismoRESUMO
Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.
Assuntos
Células Epiteliais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas c-myc/genética , Telomerase/metabolismo , Proteínas Wnt/genética , Algoritmos , Animais , Biópsia , Proliferação de Células , Procedimentos Cirúrgicos Dermatológicos , Perfilação da Expressão Gênica , Cabelo/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Imuno-Histoquímica , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação , RNA Mensageiro/metabolismo , Pele/citologia , Pele/enzimologia , Pele/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Telomerase/genética , Transcrição GênicaRESUMO
Telomeres protect chromosome ends and serve as a substrate for telomerase, a reverse transcriptase that adds DNA repeats to the telomere terminus. In the absence of telomerase, telomeres progressively shorten, ultimately leading to telomere uncapping, a structural change at the telomere that activates DNA damage responses and leads to ligation of chromosome ends. Telomere uncapping has been implicated in aging and cancer, yet the precise mechanism of uncapping and its relationship to cell cycle remain to be defined. Here, we show that telomeres uncap in an S-phase-dependent manner in gastrointestinal progenitors of TERT(-/-) mice. We develop an in vivo assay that allows a quantitative kinetic assessment of telomere dysfunction-induced apoptosis and its relationship to cell cycle. By exploiting the mathematical relationship between rates of generation and clearance of apoptotic cells, we show that 86.2 +/- 8.8% of apoptotic gastrointestinal cells undergo programmed cell death either late in S-phase or in G2. Apoptosis is primarily triggered via a signaling cascade from newly uncapped telomeres to the tumor suppressor p53, rather than by chromosome fusion-bridge breakage, because mitotic blockade did not alter the rate of newly generated apoptotic bodies. These data support a model in which rapidly dividing progenitor cells within a tissue with short telomeres are vulnerable to telomere uncapping during or shortly after telomere replication.
Assuntos
Fase S/fisiologia , Células-Tronco/fisiologia , Telômero/ultraestrutura , Animais , Apoptose , Bromodesoxiuridina , Cruzamentos Genéticos , Dano ao DNA , Fase G2 , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Knockout , Modelos Genéticos , Células-Tronco/citologia , Telomerase/deficiência , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxy-terminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.