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

终止于O-GlcNAc循环的氨基己糖信号通路参与了细胞信号转导以及转录和翻译的调控。我们试图了解O-GlcNAc依赖信号的生物学功能，并确定O-GlcNAc代谢改变是否与糖尿病和神经退行性变等人类疾病有关。转基因过表达OGT亚型在肌肉和脂肪中诱导小鼠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。这一发现表明，糖依赖信号在营养感知和2型糖尿病发病机制中具有更普遍的作用。利用反向遗传学、基因敲除等小鼠转基因模型，我们目前正在探索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，我们在小鼠中获得了OGT的敲除等位基因和亚型等位基因。OGT基因敲除动物是胚胎致死的。然而，来自这些小鼠的小鼠胚胎成纤维细胞正被用于研究胰岛素信号级联。具有OGT等位基因亚型的胚胎干细胞用于体外分化为包括胰岛β细胞在内的许多谱系。有趣的是，来自OGT亚型等位基因的Beta细胞产生的胰岛素mRNA比对照细胞多得多，这表明OGT在调节胰岛素分泌方面发挥了作用。 最近，人类O-GlcNAcase基因在墨西哥裔美国人中被鉴定为非胰岛素依赖型糖尿病(NIDDM)易感基因。我们现在已经在小鼠中定位了O-GlcNAcase基因(MGEA5)。利用组织特异性启动子驱动重组酶在不同靶组织中的表达，我们正在研究O-GlcNAcase中断对生理的影响。O-GlcNAcase在发育早期被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显示出O-GlcNAc水平的显著变化和生长缓慢。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循环可能如何影响2型糖尿病和神经退行性变中放松调控的营养感知通路。目前正在对三个模型系统进行分析：苍蝇、老鼠和线虫。 为了研究氨基己糖信号在更易遗传的生物体中的功能，我们检测了秀丽线虫中OGT和O-GlcNAcase的零等位基因，这些等位基因是活的和可育的。在线虫中，高度保守的胰岛素样信号级联调节大量营养素的储存、寿命和Dauer的形成。我们证明，OGT和OGA缺失突变体表现出显著的代谢变化，表现为海藻糖水平和糖原储存水平的上升，以及甘油三酯水平的下降。OGT基因敲除抑制了胰岛素样受体基因daf-2的温度敏感等位基因诱导的达尔幼虫的形成。OGA基因敲除促进了Dauer的形成，提示在缺乏O-GlcNAcase活性的情况下发生了胰岛素抵抗。我们的发现表明，OGT和O-GlcNAcase调节线虫的胰岛素活动，并为研究O-GlcNAc在细胞信号转导、胰岛素抵抗和肥胖中的作用提供了一个独特的遗传模型。这些研究通过检测线虫中与O-GlcNAc循环干扰相关的转录变化而得到扩展。表达芯片和染色质免疫沉淀研究都表明O-GlcNAc循环的缺陷会显著影响基因的表达。很可能这些转录变化通常与营养感知氨基己糖信号通路有关。我们的数据指出了对干细胞命运的影响，这与线虫的生殖系干细胞有关。 秀丽线虫也是研究神经退行性变的一个很好的模型系统。终止于O-GlcNAc加成的氨基己糖信号通路被认为在神经退行性变中起关键作用。在这些疾病中，聚集成聚集体的蛋白质，如tau和淀粉样前体蛋白，被O-GlcNAc严重修饰，也被磷酸化。为了研究O-GlcNAc在变态反应中的作用，我们建立了一个线虫变态反应模型，在该模型中氨基己糖信号转导的酶被系统地删除。这一策略是基于之前的工作，展示了线虫在模拟一种形式的立体病FTDP-17中的效用。我们发现，OGT-1，O-GlcNAc转移酶的缺失可以保护线虫免受tau诱导的神经病变的影响。这种保护与与聚集形成相关的tau过度磷酸化的减少有关(见图2)。这一遗传顺应性的肌萎缩侧索硬化症模型正在被用来研究去除OGT-1如何对tau诱导的神经病起到保护作用。

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. Analysis is being pursued in three model systems: fly, mouse and nematode. 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. Our data point to an impact on stem cell fate, which is linked to germline stem cells in C. elegans. 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.