近年来，RNA表观转录修饰是研究热门领域之一，N⁶-甲基腺嘌呤(N6-methyladenosion, m⁶A)是哺乳动物mRNA上丰度最高的一种表观转录修饰方式，由甲基转移酶、去甲基化酶和其结合蛋白共同调节。通过影响mRNA的代谢参与了多种生理与病理的调控。如果m⁶A修饰出现异常，可能导致肿瘤和神经退行性病变等疾病的发生。 

迄今为止的研究表明RNA m⁶A去甲基化酶ALKBH5在中枢神经系统、生殖系统发挥着重要的生理调控功能，同时也与乳腺癌、胰腺癌、结肠癌等多种肿瘤的进展过程密切相关。本课题组主要关注RNA m⁶A甲基化在脑肿瘤与神经退行性病变过程中的功能与机制研究，本课题即以ALKBH5为切入点，一方面探讨ALKBH5在脑肿瘤胶质母细胞瘤(Glioblastoma multiforme, GBM)中的促癌机制；另一方面则是ALKBH5在小脑神经退行性病病变过程中的作用。

胶质母细胞瘤是中枢神经系统最恶性的肿瘤之一，为了提高其疗效、改善患者预后，亟需对齐发病机制有更加全面和深入的了解。有研究报道铁代谢紊乱时会产生氧化应激反应，将加速胶质母细胞瘤的进展，使得患者的存活率下降，而引发铁代谢紊乱的分子调控机制仍需要进一步的研究。我们前期研究发现胰腺导管腺癌中ALKBH5通过调控铁代谢可以控制肿瘤的进展，同时有研究报道ALKBH5在胶质母细胞瘤中过表达并具有促癌作用，因此我们推测胶质母细胞瘤中ALKBH5的过表达是否也会通过m⁶A调控，导致铁代谢发生紊乱，进而促进胶质母细胞瘤的进展。

为了验证这一猜想，我们首先利用多发胶质母瘤临床病人样本进行m⁶A测序及生物信息学分析，发现肿瘤标本中铁代谢相关基因的甲基化水平出现明显变化。同时敲低ALKBH5之后，胶质母细胞瘤细胞中铁离子的含量明显减少。我们进一步利用敲低前后的胶质母细胞瘤细胞系进行m⁶A测序，发现多种铁代谢相关基因的甲基化水平发生变化，其中编码铁蛋白的FTH1 RNA甲基化水平升高最为显著。因此我们进一步探究在胶质母细胞瘤中，ALKBH5通过FTH1 RNA去甲基化对铁代谢的调控作用。我们从RNA代谢的角度分别从剪切、RNA稳定性和翻译效率三个方面探究m⁶A对FTH1靶基因的作用，发现ALKBH5对FTH1的去甲基化作用不影响其RNA稳定性和剪切，主要参与翻译调控。此外，我们发现FTH1的m⁶A位点能够影响IRP蛋白和FTH1的结合。综上，这项研究提示m⁶A去甲基化酶ALKBH5的表达异常和由此产生的m⁶A异常甲基化修饰可以通过FTH1铁代谢通路影响GBM的发生发展，从而为探究胶质母细胞瘤的发病机制与治疗靶点提供了新的思路与理论依据。

另一部分我们探讨了ALKBH5在小脑退行性病变过程中的作用。本课题组前期研究发现在低压低氧刺激下，Alkbh5缺失破坏了RNA m⁶A甲基化的平衡，导致小脑细胞异常增殖和分化，小脑发育障碍。已有研究表明m⁶A参与大脑皮层神经退行性病变过程，但是小脑退行性病变是否也受到m⁶A调控目前尚未见报道，于是我们进一步研究ALKBH5是否参与老年小鼠小脑的神经病变。我们发现随着小鼠老龄化的加剧，ALKBH5蛋白在小脑中的表达逐渐升高，且在浦肯野细胞中的表达高于其他类型的神经细胞。同时老年Alkbh5^-/-小鼠 (KO) 的体重和全脑重相较于老年野生型小鼠 (WT) 均有所下降，且老年KO小鼠的小脑重量明显减少。通过免疫组织化学实验检测中年及老年WT小鼠和KO小鼠中NeuN，Calbindin-D28K，MAP2，GFAP等蛋白的表达情况，发现老年KO小鼠的浦肯野神经元缺失且排列紊乱，同时树突数量明显减少。老年KO小鼠通过平衡木的时间相较于同龄的野生型小鼠明显增长，且出现步态不稳的情况。以上结果说明去甲基化酶ALKBH5缺失会造成老年小鼠小脑萎缩、浦肯野神经元缺失与受损，进而损害其运动协调和平衡能力，同时提示RNA m⁶A甲基化失衡会导致小脑的神经退行性病变。

综上，我们分别从两部分探究RNA m⁶A去甲基化酶ALKBH5表达异常对胶质母细胞瘤和老年小鼠小脑退行性病变的影响，该研究为治疗相关的神经系统疾病提供了新的思路。

Abstract

In recent years, RNA epigenetic transcription modification has become one of the hot research fields. N⁶-methyladenine (m⁶A) is the most abundant posttranscriptional modification on mammalian mRNAs, which is jointly regulated by methyltransferases, demethylases, and their binding proteins. It participates in various physiological and pathological regulation by influencing mRNA metabolism. This dynamic and reversible regulation is associated with the occurrence of various diseases, and abnormal m⁶A modification may lead to the occurrence of tumors and neurodegenerative diseases.

ALKBH5 is one of the major RNA m⁶A demethylases and plays multiple biological functions in diseases. As reported, ALKBH5 is involved in the progression of glioblastoma, pancreatic cancer, colon cancer and other tumors. In addition, ALKBH5 also plays important functions in the nervous system, reproductive system, etc. Our research group focuses on investigation of RNA m⁶A methylation in brain tumors and neuronal diseases. Here, we mainly focus on ALKBH5 to study its associated mechanism of tumorigenesis of glioblastoma and neurodegeneration of the mouse cerebellum.

Glioblastoma multiform (GBM) is one of the most malignant tumors in the central nervous system. In recent years, various efforts have been paid to improve its therapeutic efficiency and clinical outcomes of the patients, which requires a comprehensive understanding of its mechanism. It has been reported that when iron metabolism is disrupted, oxidative stress reactions will occur and accelerate the progression of glioblastoma and reduce the survival rate of patients. So far, detailed molecular mechanism is unclear yet and await further investigation. Previously, ALKBH5 is found to play a role in maintaining tumorigenicity of glioblastoma in m⁶A-dependent manner. On the other hand, we previously found that ALKBH5 regulates iron metabolism in pancreatic ductal adenocarcinoma. Therefore, we speculate that if overexpression of ALKBH5 in the glioblastoma may also lead to the disorder of iron metabolism, thus triggering tumor development of glioblastoma.

To verify this hypothesis, we first performed m⁶A sequencing and bioinformatics analysis on clinical patient samples of multiple glioblastomas, and found significant changes in the methylation levels of iron metabolism related genes in the tumor samples. Meanwhile, ALKBH5 knockdown caused significant decrease of the content of iron ions in glioblastoma cells. We further compared the transcriptome-wide m⁶A changes in glioblastoma cell lines before and after knockdown ALKBH5. We found that the methylation levels of various iron metabolism-related genes changed, with the most significant increase observed in ferritin-encoding FTH1 RNA methylation levels. Therefore, we further investigated the regulatory effect of ALKBH5 on iron metabolism through FTH1 RNA demethylation in glioblastoma. We investigated the effects of m⁶A on FTH1 target genes from the perspectives of RNA metabolism, including RNA splicing, RNA stability, and translation. We found that the demethylation effect of ALKBH5 on FTH1 did not affect its RNA splicing or stability, but mainly participated in translation regulation. In addition, we found that m⁶A methylation of FTH1 can enhance the binding of IRP protein to FTH1. Those results suggest that overexpression of m⁶A demethylase ALKBH5 and the resulting abnormal m⁶A methylation can affect the occurrence and development of GBM through the FTH1 iron metabolism pathway, providing new ideas and theoretical basis for exploring the novel therapeutic targets of glioblastoma.

In the second part, we investigated the effects of RNA m⁶A demethylase Alkbh5 gene knockout on cerebellar morphology and function in elderly mice. Our group previously found that under hypobaric hypoxia stimulation, the loss of Alkbh5 affects the balance of RNA m⁶A methylation, leading to impaired postnatal cerebellar development. We further investigated here whether ALKBH5 affects cerebellar neuropathy in the elderly mice. We found that with the aging of mice, the expression of ALKBH5 protein in cerebellum gradually increased, and the expression in Purkinje cell was higher than that in other types of neural cells. At the same time, the body weight and whole brain weight of elderly Alkbh5^-/- mice (KO) decreased compared to age-matched wild-type mice (WT), and the cerebellar weight of elderly KO mice was significantly reduced. We also detected the expression of NeuN, Calbindin D28K, MAP2, GFAP and other proteins in the middle-aged and elderly WT and KO mice through immunohistochemistry experiments. The number of Purkinje cell and neuron dendrites in aged KO mice decreased significantly. The time for the elderly KO mice to pass through the balance beam was significantly longer than that of the wild type mice of the same age, and their gait was unstable. However, we failed to observe similar phenotypes in the middle-aged mice. In summary, the deficiency of demethylase ALKBH5 caused cerebellar atrophy, loss and damage of Purkinje neurons in the elderly mice, thereby impairing their motor coordination and balance abilities. The above results suggest that imbalance in RNA m⁶A methylation can lead to neurodegenerative lesions in the mouse cerebellum.

In conclusion, we explored the function and mechanism of RNA m⁶A demethylase ALKBH5 in glioblastoma and cerebellar neurodegeneration from two different viewpoints, thus providing new idea for the treatment of nervous system disease.