2023-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645300XV型肝醣貯積病是一種體染色體隱性遺傳疾病，是由GYG1突變引起的。 患有這種疾病的患者表現出心肌病變，肌肉無力和多聚葡萄醣堆積的症狀。 然而，由於它的病理生理機制仍然未知，目前尚無針對該疾病的良好藥物治療。 在不同物種的研究中知道， GYG是一種醣基轉移酶，可產生寡糖引物，並透過自身醣基化合成肝醣。 然而，人類的GYG具有兩種同種型（GYG1和GYG2），如何協同工作以促進肝醣合成目前仍是未知。因此，很迫切的需要研究兩種GYG是如何促進肝醣合成的分子機制，以利找到治療XV型肝醣貯積病的治療方法。 本計畫，我們將利用人類胚胎幹細胞及其分化而來的不同譜系細胞包含心肌，骨骼肌及肝臟細胞來全面闡明1) 人類GYGs在不同譜系細胞中肝醣合成的角色，2) XV型肝醣貯積病的病理生理機制，以及3) 透過高通量藥物篩選找到治療XV型肝醣貯積病的小分子/藥物。我們的初步結果顯示GYG1是人類幹細胞中肝醣合成所必需的，剔除GYG1其細胞肝醣合成會顯著減少。有趣的是，GYG1和GYG2的雙重敲除 (Double knockout; DKO) 卻將肝醣含量恢復到與wild type (WT) 細胞一樣的量。此外，我們發現單獨剔除GYG2結果製造出比WT還更多的肝醣。值得注意地，我們發現心肌分化過程中Wnt activator會抑制GYG2的表現進而讓肝醣得以合成並產生不正常的肝醣堆積在GYG1剔除的幹細胞衍生之心臟細胞。這些結果讓我們去假設GYG2可能是合成肝醣的負調節劑。為了證明此假說，我們分別在DKO和WT細胞中過表達GYG2。令人振奮地，結果的確證明表達過多的GYG2會抑制肝醣合成，並且我們發現GYG2是透過調控肝醣合成酶 (Glycogen synthase) 的活性來調控肝醣的含量。因此，在此計畫中，我們將對GYG2是如何在各個細胞譜系中透過對肝醣合成酶的調節去調控肝醣的合成及對XV型肝醣貯積病的病理分子機制等問題全面性地去了解。最終，我們的發現不但能了解人類肝醣合成的分子機制，最重要的能針對這種疾病患者找到治療的藥物。 Glycogen storage disease type XV (GSD XV), an autosomal recessive disorder, is associated with GYG1 mutations. Patients with the disease display cardiomyopathy, muscle weakness, and polyglucosan accumulations symptoms. However, at present no good medication treatment for the disease is available as underlying pathophysiological mechanism remains unknown. Extensive study in different species has shown that GYG, encoded glycogenin 1, is a glycosyltransferase that generates an oligosaccharide primer for glycogen synthesis by autoglucosylation. However, humans carry two GYG isoforms (GYG1 and GYG2), and how they work in concert to facilitate glycogen synthesis remains elusive. It is compelling needed to investigate the molecular mechanism of glycogen synthesis facilitated by two GYGs in order to find a better way to treat human patients with the disease. In this proposal, we will take advantage of human embryonic stem cells (hESCs) and their derived cell lineages, including cardiomyocyte, skeletal muscle and hepatocyte to comprehensively elucidate (1) the role of human GYGs in glycogen synthesis of different cell lineages, (2) the pathophysiological mechanism of glycogen storage disease type XV (GSD XV), and (3) to ultimately identify small molecules/drugs that can treat GSD XV. Our preliminary studies showed that the function of GYG1 is crucial for glycogen synthesis in hESCs, as evidenced by a dramatic reduction of glycogen in GYG1 single knockout mutant. Interestingly, in the GYG1 and GYG2 dual knockout cell lines, glycogen was restored to a comparable level to that in WT. Moreover, we found that GYG2 single knockout line showed more glycogen production than WT. Notably, Wnt signaling inhibited GYG2 expression, and thereby, glycogen could be re-synthesized and cause polyglucosan body accumulation in GYG1-/--derived cardiomyocytes. Altogether, these results lead us to hypothesize that GYG2 functions as a negative regulator of cellular glycogen content. To test this hypothesis, we next ectopically expressed GYG2 in DKO and WT lines and found that glycogen synthesis was shut down in both cell lines, which further suggested that GYG2 is a negative regulator of glycogen. Notably, we further found that GYG2 regulated glycogen content through modulating glycogen synthase activity. Therefore, we propose to examine 1) how GYG2 regulates glycogen synthase activity to modulate glycogen content; 2) the molecular mechanisms underlying GYG1 deficiency-induced GSD XV; and 3) to identify small molecules that can rescue the phenotypes using a high throughput chemical screening. Ultimately, our discoveries here will pave the way to find better treatment for patients with this disease.人類多能幹細胞;肝醣;肝醣合成;肝醣貯積病XV;human pluripotent stem cells;glycogenin;glycogen synthesis; glycogen storage disease XV