Data_Sheet_1_Suppression of hnRNP A1 binding to HK1 RNA leads to glycolytic dysfunction in Alzheimer’s disease models.zip

ObjectiveTo investigate the mechanism of RNA-binding protein hnRNP A1 in mouse hippocampal neurons (HT22) on glycolysis.
MethodsRIP and CLIP-qPCR were performed by HT22 in vitro to observe the mechanism of hnRNP A1 regulating the expression of key proteins in glycolysis. The RNA binding domain of hnRNP A1 protein in HT22 was inhibited by VPC-80051, and the effect of hnRNP A1 on glycolysis of HT22 was observed. Lentivirus overexpression of hnRNP A1 was used to observe the effect of overexpression of hnRNP A1 on glycolysis of Aβ25–35-injured HT22. The expression of hnRNP A1 in brain tissues of wild-type mice and triple-transgenic (APP/PS1/Tau) AD mice at different ages was studied by Western blot assay.
ResultsThe results of RIP experiment showed that hnRNP A1 and HK1 mRNA were significantly bound. The results of CLIP-qPCR showed that hnRNP A1 directly bound to the 2605-2821 region of HK1 mRNA. hnRNP A1 inhibitor can down-regulate the expression of HK1 mRNA and HK1 protein in HT22 cells. Overexpression of hnRNP A1 can significantly reduce the toxic effect of Aβ25–35 on neurons via the hnRNP A1/HK1/ pyruvate pathway. In addition, inhibition of hnRNP A1 binding to amyloid precursor protein (APP) RNA was found to increase Aβ expression, while Aβ25–35 also down-regulated hnRNP A1 expression by enhancing phosphorylation of p38 MAPK in HT22. They interact to form bidirectional regulation, further down-regulating the expression of hnRNP A1, and ultimately aggravating glycolytic dysfunction. Protein immunoblotting showed that hnRNP A1 decreased with age in mouse brain tissue, and the decrease was greater in AD mice, suggesting that the decrease of hnRNP A1 may be a predisposed factor in the pathogenesis of AD.

研究目的：探究RNA结合蛋白（RNA-binding protein）异质性核核糖核蛋白A1（hnRNP A1）在小鼠海马神经元HT22细胞中对糖酵解的调控机制。
实验方法：采用体外培养的HT22细胞进行RNA免疫沉淀（RIP）与交联免疫沉淀定量PCR（CLIP-qPCR）实验，以观察hnRNP A1对糖酵解关键蛋白表达的调控机制。使用VPC-80051抑制HT22细胞中hnRNP A1蛋白的RNA结合结构域，观察hnRNP A1对HT22细胞糖酵解的影响。采用慢病毒介导hnRNP A1过表达，观察过表达hnRNP A1对Aβ₂₅₋₃₅损伤的HT22细胞糖酵解的影响。采用蛋白质印迹法（Western blot）检测不同年龄野生型小鼠与三转基因（APP/PS1/Tau）阿尔茨海默病（AD）小鼠脑组织中hnRNP A1的表达水平。
实验结果：RNA免疫沉淀实验结果显示，hnRNP A1与己糖激酶1（HK1）mRNA存在显著结合。交联免疫沉淀定量PCR实验结果表明，hnRNP A1可直接结合于HK1 mRNA的2605-2821区域。hnRNP A1抑制剂可下调HT22细胞中HK1 mRNA与HK1蛋白的表达水平。过表达hnRNP A1可通过hnRNP A1/HK1/丙酮酸通路显著减轻Aβ₂₅₋₃₅对神经元的毒性作用。此外，研究发现抑制hnRNP A1与淀粉样前体蛋白（APP）RNA的结合可上调Aβ的表达；而Aβ₂₅₋₃₅也可通过增强HT22细胞中p38丝裂原活化蛋白激酶（p38 MAPK）的磷酸化水平，下调hnRNP A1的表达。二者相互作用形成双向调控，进一步下调hnRNP A1的表达，最终加剧糖酵解功能障碍。蛋白质印迹实验结果显示，小鼠脑组织中hnRNP A1的表达随年龄增长而下调，且在AD小鼠中下调幅度更为显著，提示hnRNP A1表达下调可能是阿尔茨海默病发病的易感因素之一。