E3 泛素连接酶 Siah-1 在高糖和低氧条件下下调突触素的表达。
E3 ubiquitin ligase Siah-1 downregulates synaptophysin expression under high glucose and hypoxia.
机构信息
Department of Neurology, The 10th People's Hospital, Tongji University 301# Middle Yanchang Road, Shanghai 200072, China.
Department of Neurology, Huashan hospital, State Key Laboratory of Medical Neurobiology, Fudan University 12# Middle Wulumuqi Road, Shanghai, 200040 China.
出版信息
Am J Transl Res. 2015 Jan 15;7(1):15-27. eCollection 2015.
BACKGROUND
Diabetes is proved to be one of the independent risk factors for cognitive dysfunction. The pathophysiologic changes caused by diabetes including hyperglycemia and tissue hypoxia may contribute greatly to cognitive decline. In the present study, we demonstrate E3 Ubiquitin Ligase Siah-1 downregulates the key synaptic protein Synaptophysin expression under high glucose and hypoxia condition which may be the underlying factor leading to cognitive dysfunction in diabetic patients.
METHODS
In this study, hypoxia (2% oxygen) and high glucose (50 mM) were used to treat primary neuronal culture. By using quantitative PCR and western blotting we determined the influence of hypoxia and high glucose on the expression of synaptophysin and Siah-1 and the phosphorylated forms of extracellular signal-regulated kinase (ERK). Knockdown of Siah-1, inhibitors for proteasome, lysosome and ERK kinase was employed to evaluate the role of Siah-1 and ERK activity on the expression of synaptophysin. By immunoprecipitation we also examined the role of Siah-1 in the ubiquitination of synaptophysin under hypoxic and hyperglycemic condition.
RESULTS
We demonstrated that hypoxia and high glucose together but not hypoxia or high glucose along mediated posttranscriptional reduction of synaptophysin with increased ERK phosphorylation and Siah-1 expression. The downregulation of synaptophysin was reversed by inhibition of ERK and Siah-1 knockdown. Overexpression of Siah-1 accelerated the degradation of synaptophysin under hypoxia and high glucose conditions and promoted the ubiquitination of synaptophysin.
CONCLUSIONS
The present results demonstrate that Siah-1 is the key factor that contributes to hypoxia and high glucose mediated synaptophysin degradation.
背景
糖尿病被证实是认知功能障碍的独立危险因素之一。糖尿病引起的病理生理变化，包括高血糖和组织缺氧，可能极大地导致认知能力下降。在本研究中，我们证明 E3 泛素连接酶 Siah-1 在高糖和缺氧条件下下调关键突触蛋白突触小泡蛋白的表达，这可能是导致糖尿病患者认知功能障碍的潜在因素。
方法
在这项研究中，使用缺氧（2%氧气）和高糖（50mM）处理原代神经元培养物。通过定量 PCR 和 Western blot，我们确定了缺氧和高糖对突触小泡蛋白和 Siah-1以及细胞外信号调节激酶（ERK）磷酸化形式表达的影响。使用 Siah-1 的小干扰 RNA（siRNA）、蛋白酶体、溶酶体和 ERK 激酶抑制剂来评估 Siah-1 和 ERK 活性对突触小泡蛋白表达的作用。通过免疫沉淀，我们还研究了 Siah-1 在缺氧和高糖条件下突触小泡蛋白泛素化中的作用。
结果
我们证明，缺氧和高糖共同作用而非单独作用于缺氧或高糖，介导了突触小泡蛋白的转录后减少，同时伴随着 ERK 磷酸化和 Siah-1 表达增加。ERK 抑制和 Siah-1 敲低逆转了突触小泡蛋白的下调。在缺氧和高糖条件下，过表达 Siah-1 加速了突触小泡蛋白的降解，并促进了突触小泡蛋白的泛素化。
结论
本研究结果表明，Siah-1 是导致缺氧和高糖介导的突触小泡蛋白降解的关键因素。
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