Targeted disruption the enzymes of O-GlcNAc cycling: Animal models of Disease

靶向破坏 O-GlcNAc 循环酶：疾病动物模型

• 批准号: 7593744
• 负责人: John A. Hanover
• 金额: $ 38.26万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593744
• 关键词: Alleles; Amyloid beta-Protein Precursor; Animal Disease Models; Beta Cell; Biological Models; Biological Process; Caenorhabditis elegans; Cell Death; Cells; Data; Defect; Development; Diabetes Mellitus; Disease; Disruption; Elevation; Embryo; Employee Strikes; Enzymes; Excision; Exhibits; FTD with parkinsonism; Fatty acid glycerol esters; Fibroblasts; Gene Expression; Genes; Genetic Models; Glycogen; Goals; Growth; Hexosamines; Human; In Vitro; Insulin; Insulin Resistance; Knock-out; Larva; Leptin; Link; Longevity; Macronutrients Nutrition; Mediating; Messenger RNA; Metabolic; Metabolism; Mexican Americans; Modeling; Molecular; Mus; Muscle; Nematoda; Nerve Degeneration; Neurodegenerative Disorders; Neuropathy; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Nutrient; O-GlcNAc transferase; Obesity; Organism; Pathogenesis; Pathway interactions; Physiological; Play; Polysaccharides; Predisposition; Protein Isoforms; Protein Overexpression; Proteins; Receptor Gene; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Structure of beta Cell of islet; Tauopathies; Technology; Temperature; Tissues; Transcriptional Regulation; Transgenic Mice; Transgenic Model; Transgenic Organisms; Translations; Trehalose; Triglycerides; Work; base; blastomere structure; chromatin immunoprecipitation; detection of nutrient; embryonic stem cell; fly; gain of function; human disease; insulin secretion; insulin signaling; knockout animal; mutant; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; positional cloning; promoter; protective effect; recombinase; tau Proteins

The hexosamine signaling pathway terminating in O-GlcNAc cycling has been implicated in cellular signaling cascades and regulation of transcription and translation. We seek to understand the biological functions of O-GlcNAc-dependent signaling and to determine whether altered O-GlcNAc metabolism contributes to human diseases such as diabetes mellitus and neurodegeneration. Transgenic overexpression of an isoform of OGT in muscle and fat induced Insulin resistance and Hyperleptinemia in mice. These data demonstrate a central role for OGT in the insulin and leptin-signaling cascades. The findings suggest a more general role for glycan-dependent signaling in nutrient sensing and the pathogenesis of type-2 diabetes. Using reverse genetics, knockout, and other mouse transgenic models, we are currently exploring the role of the enzymes of O-GlcNAc metabolism in signal transduction and the pathogenesis of diabetes mellitus. Using cre/lox technology, we have made knockout and hypomorphic alleles of OGT in the mouse. OGT knockout animals are embryonic lethal. However, mouse embryo fibroblasts derived from these mice are being used to examine the insulin signaling cascade. Embryonic stem cells with a hypomorphic OGT allele are used for in vitro differentiation into a number of lineages including the pancreatic Beta cells. Interestingly, Beta cells derived from the hypomorphic OGT allele produce much more insulin mRNA than control cells suggesting a role for OGT in regulating insulin secretion. Recently, the human O-GlcNAcase gene was identified as a non-insulin dependent diabetes mellitus (NIDDM) susceptibility locus in Mexican Americans. We have now targeted the O-GlcNAcase gene (MGEA5) in the mouse. Using tissue specific promoters to drive expression of cre-recombinase in various target tissues, we are examining the physiological impact of O-GlcNAcase disruption. Knockout of O-GlcNAcase during early development leads to embryonic cell death. Fibroblasts derived from these knockout animals show dramatically altered O-GlcNAc levels and slower growth. Tissue-specific disruptions of the O-GlcNAcase gene are in progress. Our goal is to understand how interference with O-GlcNAc cycling may impact nutrient sensing pathways deregulated in type-2 diabetes and neurodegeneration. To examine the function of hexosamine signaling in a more genetically amenable organism, we have examined null alleles of OGT and the O-GlcNAcase in Caenorhabditis elegans that are viable and fertile. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity and dauer formation. We demonstrate that the OGT and OGA null mutants exhibit striking metabolic changes manifested in an elevation in trehalose levels and glycogen stores with a concomitant decrease in triglycerides levels. The OGT knockout suppresses dauer larvae formation induced by a temperature sensitive allele of the insulin-like receptor gene daf-2. The OGA knockout enhances dauer formation suggesting the development of insulin resistance in the absence of O-GlcNAcase activity. Our findings demonstrate that OGT and O-GlcNAcase modulate insulin action in C. elegans and provide a unique genetic model for examining the role of O-GlcNAc in cellular signaling, insulin resistance and obesity. These studies have been extended by examining the transcriptional changes associated with interference of O-GlcNAc cycling in C. elegans. Both expression microarrays and chromatin immunoprecipitation studies argue that defects in O-GlcNAc cycling dramatically impact gene expression. It is likely that these transcriptional changes are normally linked to the nutrient sensing hexosamine-signaling pathway. Caenorhabditis elegans is also an excellent model system in which to examine neurodegeneration. The hexosamine signaling pathway terminating in O-GlcNAc addition has been proposed to play a key role in neurodegeneration. In these disorders, the proteins accumulating as aggregates such as tau and amyloid precursor protein are heavily modified with O-GlcNAc and are also phosphorylated. To examine the role of O-GlcNAc in tauopathy we have developed a C. elegans model of tauopathy in which the enzymes of hexosamine signaling have been systematically deleted. This strategy is based on previous work demonstrating the utility of C. elegans in modeling the one form of tauopathy, FTDP-17. We find that the loss of OGT-1, the O-GlcNAc transferase protects the nematode from human tau-induced neuropathy. This protection is associated with a decrease in the hyperphosphorylation of tau associated with aggregate formation (See Figure 2). This genetically amenable model of tauopathy is being exploited to examine how removal of OGT-1 exerts its protective effect on tau-induced neuropathy.

终止于 O-GlcNAc 循环的己糖胺信号通路与细胞信号级联以及转录和翻译的调节有关。我们试图了解 O-GlcNAc 依赖性信号传导的生物学功能，并确定 O-GlcNAc 代谢的改变是否会导致糖尿病和神经退行性疾病等人类疾病。肌肉和脂肪中 OGT 亚型的转基因过度表达可诱导小鼠出现胰岛素抵抗和高瘦素血症。这些数据证明了 OGT 在胰岛素和瘦素信号级联中的核心作用。研究结果表明，聚糖依赖性信号传导在营养感知和 2 型糖尿病发病机制中具有更普遍的作用。我们目前正在利用反向遗传学、基因敲除和其他小鼠转基因模型，探索O-GlcNAc代谢酶在信号转导和糖尿病发病机制中的作用。 利用cre/lox技术，我们在小鼠中制作了OGT的敲除和亚等位基因。 OGT 基因敲除动物胚胎是致命的。 然而，源自这些小鼠的小鼠胚胎成纤维细胞被用来检查胰岛素信号级联。具有亚效型 OGT 等位基因的胚胎干细胞可在体外分化为多种谱系，包括胰腺 Beta 细胞。 有趣的是，源自低效 OGT 等位基因的 Beta 细胞比对照细胞产生更多的胰岛素 mRNA，表明 OGT 在调节胰岛素分泌中发挥作用。 最近，人类 O-GlcNAcase 基因被鉴定为墨西哥裔美国人的非胰岛素依赖型糖尿病 (NIDDM) 易感基因座。 我们现在已经针对小鼠体内的 O-GlcNAcase 基因 (MGEA5) 进行了靶向治疗。 使用组织特异性启动子驱动 cre 重组酶在各种靶组织中的表达，我们正在研究 O-GlcNAcase 破坏的生理影响。 早期发育过程中 O-GlcNAcase 的敲除会导致胚胎细胞死亡。 来自这些基因敲除动物的成纤维细胞表现出显着改变的 O-GlcNAc 水平和更慢的生长。 O-GlcNAcase 基因的组织特异性破坏正在进行中。我们的目标是了解干扰 O-GlcNAc 循环如何影响 2 型糖尿病和神经退行性疾病中营养传感途径的失调。 为了检查己糖胺信号传导在遗传上更顺从的生物体中的功能，我们检查了具有活力和繁殖力的秀丽隐杆线虫中 OGT 和 O-GlcNAcase 的无效等位基因。在线虫中，高度保守的类胰岛素信号级联调节大量营养素的储存、寿命和多尔形成。我们证明 OGT 和 OGA 无效突变体表现出显着的代谢变化，表现为海藻糖水平和糖原储存升高，同时甘油三酯水平降低。 OGT 敲除可抑制由胰岛素样受体基因 daf-2 的温度敏感等位基因诱导的 dauer 幼虫形成。 OGA 敲除增强了 dauer 形成，表明在缺乏 O-GlcNAcase 活性的情况下会出现胰岛素抵抗。我们的研究结果表明，OGT 和 O-GlcNAcase 调节线虫中的胰岛素作用，并为检查 O-GlcNAc 在细胞信号传导、胰岛素抵抗和肥胖中的作用提供了独特的遗传模型。 通过检查线虫中与 O-GlcNAc 循环干扰相关的转录变化，这些研究得到了扩展。 表达微阵列和染色质免疫沉淀研究都认为 O-GlcNAc 循环的缺陷会极大地影响基因表达。 这些转录变化通常可能与营养感应己糖胺信号通路有关。 秀丽隐杆线虫也是检查神经退行性变的优秀模型系统。己糖胺信号通路终止于 O-GlcNAc 添加，已被认为在神经退行性变中发挥关键作用。在这些疾病中，以聚集体形式积累的蛋白质（例如 tau 蛋白和淀粉样前体蛋白）经过 O-GlcNAc 的大量修饰，并且也被磷酸化。为了研究 O-GlcNAc 在 tau 蛋白病中的作用，我们开发了 tau 蛋白病的秀丽隐杆线虫模型，其中己糖胺信号转导酶已被系统删除。该策略基于先前的工作，证明了秀丽隐杆线虫在模拟一种 tau 蛋白病 FTDP-17 方面的效用。我们发现 OGT-1（O-GlcNAc 转移酶）的缺失可以保护线虫免受人类 tau 诱导的神经病变。这种保护作用与聚集体形成相关的 tau 蛋白过度磷酸化的减少有关（见图 2）。这种适合基因的 tau 病模型被用来研究 OGT-1 的去除如何对 tau 诱导的神经病发挥保护作用。