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

终止于O-GlcNAc循环的己糖胺信号传导途径涉及细胞信号传导级联以及转录和翻译的调节。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代谢的改变是否有助于人类疾病，如糖尿病和神经变性。肌肉和脂肪中OGT亚型的转基因过表达诱导小鼠的胰岛素抵抗和高瘦素血症。这些数据表明OGT在胰岛素和瘦素信号级联中的核心作用。这些发现表明，聚糖依赖性信号在营养感知和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、基因敲除和其他小鼠转基因模型，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，我们在小鼠中制备了OGT的敲除和亚型等位基因。OGT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被用于检查胰岛素信号级联。具有亚型OGT等位基因的胚胎干细胞用于体外分化成包括胰腺β细胞在内的许多谱系。 有趣的是，来自亚型OGT等位基因的β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的作用。我们正在探索干细胞生物学的各个方面，利用我们生成的遗传模型。 最近，人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。 我们现在已经在小鼠中靶向了O-GlcNAcase基因（MGEA 5）。 使用组织特异性启动子来驱动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。 在早期发育过程中敲除O-GlcNAc酶导致胚胎细胞死亡。 来源于这些敲除动物的成纤维细胞显示出显著改变的O-GlcNAc水平和较慢的生长。脑中O-GlcNAc酶的敲除导致神经发生和垂体发育的缺陷。O-GlcNAcase基因的其他组织特异性破坏正在进行中。我们的目标是了解O-GlcNAc循环的干扰如何影响2型糖尿病和神经退行性变中失调的营养感知途径。 目前正在三种模式系统中进行分析：苍蝇、小鼠和线虫。 为了研究己糖胺信号传导在遗传上更适合的生物体中的功能，我们已经研究了秀丽隐杆线虫中OGT和O-GlcNAc酶的无效等位基因，这些等位基因是可行的和可育的。在线虫中，一个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形成。我们证明，OGT和OGA无效突变体表现出惊人的代谢变化，表现在海藻糖水平和糖原储备的升高，伴随着甘油三酯水平的降低。OGT敲除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dauer形成，表明在O-GlcNAc酶活性不存在的情况下胰岛素抗性的发展。我们的研究结果表明OGT和O-GlcNAc酶调节C.并提供了一种独特的遗传模型，用于检查O-GlcNAc在细胞信号传导、胰岛素抵抗和肥胖症中的作用。 这些研究已经通过检查与C.优雅 表达微阵列和染色质免疫沉淀研究都认为O-GlcNAc循环的缺陷会显著影响基因表达。 很可能这些转录变化通常与营养传感己糖胺信号通路有关。我们的数据指向对干细胞命运的影响，这与蠕虫中的生殖系干细胞有关。 秀丽隐杆线虫也是研究神经变性的一个很好的模型系统。已提出终止于O-GlcNAc加成的己糖胺信号传导途径在神经变性中起关键作用。在这些疾病中，聚集为聚集体的蛋白质如tau和淀粉样前体蛋白被O-GlcNAc严重修饰，并且也被磷酸化。为了研究O-GlcNAc在tau蛋白病中的作用，我们开发了一种C.这是tau蛋白病的elegans模型，其中己糖胺信号传导的酶已被系统地删除。这个策略是基于以前的工作，证明了C的效用。elegans在模拟一种形式的tau蛋白病FTDP-17中的作用。我们发现，OGT-1，O-GlcNAc转移酶的损失保护线虫免受人类tau诱导的神经病变。这种保护作用与减少与聚集体形成相关的tau过度磷酸化有关。这种遗传上适合的tau蛋白病模型正在被用来研究OGT-1的去除如何对tau诱导的神经病变发挥其保护作用。我们也使用了C。优雅的遗传学探索氧化损伤反应途径和转录反应的氧化损伤。我们的研究结果表明O-GlcNAc循环在氧化损伤下游的DNA损伤修复途径中的作用。 我们在小鼠、苍蝇和蠕虫中的研究强烈表明，O-GlcNAc循环的酶在信号传导、通过多梳和三胸复合物的表观遗传调节以及调节线粒体/基因组相互作用中发挥重要作用。 因此，它是包括代谢综合征、阿尔茨海默病和癌症在内的衰老疾病的潜在重要的未开发的治疗靶点。 这些疾病的探针正在开发，依赖于这些动物模型中开发的技术。

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. We are exploring various aspects of stem cell biology exploiting genetic models we have generated. 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. Knockout of O-GlcNAcase in the brain leads to defects in neurogenesis and pituitary development. Other 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 the worm. 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. This genetically amenable model of tauopathy is being exploited to examine how removal of OGT-1 exerts its protective effect on tau-induced neuropathy. We have also used C. elegant genetics to explore oxidative damage response pathways and the transcriptional response to oxidative insult. Our findings suggest a role for O-GlcNAc cycling in the DNA damage repair pathway downstream of oxidative damage. Our studies in Mouse, Fly and worm strongly suggest that the enzymes of O-GlcNAc cycling perform essential functions in signaling, epigenetic regulation through the polycomb and trithorax complexes and in modulating mitochondrial/genome interactions. It is therefore a potentially important unexplored therapeutic target for diseases of aging including metabolic syndrome, Alzheimer's disease and cancer. Probes for these diseases are being developed that rely on the technologies developed in these animal models.