轻度认知功能障碍(MCI)中铁代谢异常对BACE1酶切位点选择的影响及机制研究

Mild cognitive impairment (MCI) is a transitional stage between normal aging and dementia. Identification of MCI and its underlying pathologic mechanisms can help find target for better use of therapeutic interventions. Our previous clinical study showed that iron metabolism markers such as anemia and ferritin are associated with cognitive dysfunction. Moreover, it has been confirmed that abnormal iron metabolism and ferroptosis in the brain play a key role in the dementia pathology. β-site APP cleaving enzyme 1 (BACE1) is a rate limiting enzyme for amyloid β precursor protein (APP) processing and amyloid β protein (Aβ) production. We found that some external factors could regulate BACE1 cleavage site selection for APP protein, resulting in higher Aβ production and dementia progression. Therefore, in this study, we will explore the diagnostic value of abnormal iron metabolism for cognitive dysfunction in clinical, pathologic and molecular level. Besides, we will explore the effect of abnormal iron metabolism on BACE1 activity and cleavage site selection by using metal chelating BACE1 inhibitors. Our study will provide new evidence for the underlying mechanism in MCI and promising intervention in the future.

轻度认知功能障碍患者(MCI)是发生痴呆的高危人群，识别MCI和研究其发生机制和干预措施，有助于发现最佳干预时机。我们前期临床研究表明贫血、铁蛋白等铁代谢异常相关指标和脑血管疾病预后及认知功能障碍有潜在联系；而脑内铁代谢异常、铁死亡等已证实在痴呆的发病机理中起了重要作用。BACE1是APP酶切和Aβ生成过程的限速酶，申请人研究表明外部因素可以通过改变BACE1在APP蛋白酶切位点的改变促进Aβ生成和痴呆的发生。本研究假设，铁代谢异常通过改变BACE1分泌酶在APP蛋白主要酶切位点的选择，影响Aβ的产生，进而促进认知功能障碍的发生。我们拟临床、细胞及动物研究，从临床上验证铁代谢异常对认知障碍的诊断价值，从细胞动物模型上明确铁代谢异常对BACE1活性及酶切位点改变的作用和机制，并探讨金属螯合BACE1抑制剂干预的作用，为MCI的诊断及干预提供新的证据。

BACE1对Aβ生成有重要意义，而临床药物研究中BACE1抑制剂虽然可以显著降低Aβ的生产，但是并没有改善AD患者的认知障碍。我们主要研究了BACE1、铁死亡、和认知障碍之间的联系，探索是否可以通过调控BACE1和铁死亡，改善AD小鼠的认知障碍。我们研究表明，BACE1和铁代谢、铁死亡通路相关；铁死亡抑制可能能够缓解BACE1抑制导致的认知功能障碍；轻度认知功能障碍(MCI)人群中，BACE1相关血管障碍更为严重，但是BACE1相关铁代谢产物血液水平未见显著差别。结果发现，增加细胞内铁水平后BACE1的表达显增加(127%±23.3, P =0.0055)；抑制BACE1活性和表达导致胞内的Fe2+浓度显著增加; 抑制BACE1后细胞内ACSL4的表达显著升高(67.25%± 0.134；P=0.0055)，GPX4的表达显著减低(55.39%±0.08462, P=0.0007)，铁蛋白的表达显著降低(-37.69% ±0.09332, P=0.0033); 在小鼠模型中，BACE1抑制剂和海马区定位AAV8-BACE1 KO病毒注射作用一致，减少了海马区PSD95表达(-31.34% ± 0.07654, P=0.0027; -29.63% ± 0.06905, P =0.0078)以及树突棘密度(-8.295% ± 0.02558, P =0.0316)，同时在如新事物识别 (-47.05%±0.2031, P=0.044 vs.-39.06%±0.1151, P=0.0035)等行为学检测中表现更差；在队列研究中，同正常人群比较，MCI人群的BACE1相关微血管参数DCP (deep capillary plexus;0.86±0.06vs. 0.72±0.16mm2;P<0.001)choriocapillaris (2.83±0.08vs.2.53±0.24 mm2;P<0.001) 均有显著下降，但是外周血中转铁蛋白(2.53±0.38vs.2.46±0.36g/L;P=0.138)和铁蛋白(252.94±187.05 vs.260.18±198.43ng/mL;P=0.775) 水平未见显著差异。综上，我们的研究成果提示可以通过干预铁死亡通路，减轻BACE1 抑制剂对神经系统的毒性，极大改善BACE1 抑制剂在阿尔茨海默病的应用前景。

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