CaMKⅡ介导内质网应激通路促X-连锁肾上腺脑白质营养不良（X-ALD）发生的机制研究

X-linked adrenoleukodystrophy is a rare inherited disorder due to the deficiency of adrenoleukodystrophy protein (ALDP) that results in very long chain fatty acids (VLCFA) accumulation in various tissues, with adrenal insufficiency and cerebral inflammatory degeneration being the main manifestation. Toxic effects of VLCFA on inducing glial cells and neurons apoptosis are important factors to trigger cerebral degeneration. Currently reports have showed that VLCFA could induce endoplasmic reticulum (ER) stress and apoptosis, targeting ER stress-associated apoptosis may serve as a potent approach to prevent cerebral degeneration. Our previous studies showed that saturated VLCFA (C26:0) could promote the phosphorylation of CaMKⅡ, increase the expression of ER stress markers (CHOP) and trigger apoptosis in the 293T cells with siRNA-mediated ABCD1 knockdown, but not in controls. Furthermore, inhibition of CaMKⅡphosphorylation caused a significant decrease in the rate of apoptosis in ABCD1‑silenced 293T cells. Hence, we put forward a viewpoint that CaMKⅡplays a crucial role in the ER stress related pathway caused by VLCFA toxicity. In this proposal, we will use X-ALD mouse model to investigate the expression of CaMKⅡand other associated markers of ER stress. Meanwhile, we will compare pathological changes in brain and spinal cord tissues, and the cognitive and locomotor impairment of X-ALD mouse model before and after the exposure to CaMKⅡinhibitors. This study is expected to reveal the underlying mechanism of X-ALD neurodegeneration caused by ER stress-mediated apoptosis via CaMKⅡphosphorylation, and provide theoretical basis and potential targets for X-ALD treatment.

X-ALD是致残致死的罕见遗传病，表现为神经退行性病变及肾上腺皮质功能减退。ALD蛋白功能缺陷使极长链脂肪酸（VLCFA）异常堆积是直接致病因素；神经细胞凋亡是中枢病损的重要成因。最新研究显示VLCFA脂毒性可致细胞内质网应激(ERS)并触发凋亡，故研究作用于ERS通路的靶分子对干预神经退行性病变意义重大。预实验发现VLCFA脂毒性激活CaMKⅡ磷酸化，上调ERS信号CHOP表达，触发细胞凋亡；抑制CaMKⅡ磷酸化可减少细胞凋亡率。据此，我们提出磷酸化CaMKⅡ在脂毒性致ERS通路中发挥重要作用的假说。本项目拟采用CRISPR/Cas9构建ABCD1基因敲除鼠模型，测定神经组织CaMKⅡ表达，应用CaMKⅡ抑制剂，分析干预前后大脑和脊髓病理以及小鼠运动功能的变化，深层次揭示CaMKⅡ介导VLCFA脂毒性致ERS的原因及其参与X-ALD神经退行性病变的机制，为治疗X-ALD提供潜在新靶点。

X-连锁肾上腺脑白质营养不良被收入《第一批罕见病目录》，其诊断与治疗受到罕见病研究团队的关注。该病是由于ABCD1基因突变导致ALDP蛋白功能缺陷，使极长链脂肪酸(VLCFA)不能转膜进入过氧化物酶体进行β-氧化，在大脑、脊髓、肾上腺、睾丸等组织内过度堆积，继而出现中枢神经系统受损及肾上腺皮质功能减退的表现。临床上以儿童脑型、脊髓神经病型常见，预后差，可致残致死。因其中枢神经系统退行性病变发病机制原因不明，故尚无有效治疗方法，这为患者家庭与社会带来沉重负担。已有研究提示，神经细胞凋亡是中枢病损的重要成因，而VLCFA脂毒性可致细胞内质网应激(ERS)并触发细胞凋亡，故本研究重点在于探索ERS通路的靶分子对干预神经退行性病变的作用机制。本课题获得以下研究结果：①成功获得X-ALD患者的皮肤成纤维细胞，为后续研究X-ALD致病机制以及构建筛选与开发治疗该疾病奠定了细胞平台基础；② 通过对X-ALD患者皮肤成纤维细胞进行实验，证实了VLCFA脂毒性可触发ALD细胞内发生内质网应激，线粒体氧化应激，使线粒体内钙向细胞内流动，导致细胞内钙超载；③ 同时证实，细胞内 Ca2+浓度上升，可以提高 CaMKⅡ的磷酸化，磷酸化 CaMKⅡ是参与 ERS 的关键调控因子。④本实验还构建了ABCD1基因缺陷的小鼠，通过动物实验，发现病鼠脊髓细胞存在内质网肿胀、数量减少，与野生型小鼠脊髓组织比较，凋亡途径表达基因存在差异性，这为后续进行该途径靶基因药物治疗奠定了基础。

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