Supplementary Material for: Human apolipoprotein E ε4 allele modulates energy substrate availability, seizure burden, mortality and hippocampal injury, cell death and inflammation after neonatal hypoxic-ischemic brain injury.

Introduction: Human apolipoprotein E allele ε4 (ApoE4) is the strongest genetic risk factor for some forms of adulthood neurodegeneration linked to energetic disturbances and inflammation. We hypothesized that ApoE4 also influences neonatal brain neurodegeneration after a hypoxic ischemic (HI) insult, resulting in energy substrates (i.e., glucose, ketone bodies) disturbances, hippocampal injury, cell death and inflammation. Methods: Right-sided brain HI was induced at P10 in wild type (wt, C57BL6) and humanized ApoE3 and ApoE4 mice with sham anesthesia-exposed littermates as controls. Seizure-like activity, survival, blood glucose (BG), and ketone bodies (KB) were determined immediately after the HI insult. The hippocampi were assessed 24h and 72h after the HI insult for residual volume, cell death (α-fodrin breakdown), inflammatory markers, and transcriptomics (RNAseq). Results: Wt, ApoE3 and ApoE4 mice were congenic (>99.8% transcriptome similarity). Female ApoE4 mice had worse seizures, lower survival and smaller residual hippocampal volumes than the ApoE3 mice. All three strains had lower BG after HI. ApoE4 mice also had lower KB. Low BG was associated with higher pro-inflammatory and cell death markers in the hippocampus in all HI genotype groups at 24h but more robustly in ApoE4 mice, and in combination with high KB, was strongly linked to cell death (greater α-fodrin breakdown). Conclusion: Humanized ApoE4, compared to ApoE3, causes greater hippocampal injury, cell death and inflammation after a neonatal HI insult in association with low BG and underutilized KB. The mechanisms behind these associations need further investigation.引言：载脂蛋白E ε4等位基因（Apolipoprotein E ε4, ApoE4）是与能量代谢紊乱及炎症反应相关的成人期部分神经退行性疾病的最强遗传风险因子。本研究假设，ApoE4同样会影响缺氧缺血（hypoxic ischemic, HI）损伤后的新生小鼠脑神经退行性病变，进而引发能量底物（即葡萄糖、酮体）代谢紊乱、海马体损伤、细胞死亡及炎症反应。 方法：于出生后第10天（P10），在野生型（wild type, wt，C57BL6）以及人源化ApoE3、ApoE4小鼠中构建右侧大脑缺氧缺血模型，以接受假手术麻醉处理的同窝幼鼠作为对照。于缺氧缺血损伤后即刻检测癫痫样活动、生存率、血糖（blood glucose, BG）及酮体（ketone bodies, KB）水平。分别于损伤后24小时与72小时，对海马体开展如下评估：剩余体积、细胞死亡情况（α-血影蛋白降解产物）、炎症标志物水平，以及转录组学分析（RNA测序, RNAseq）。 结果：野生型、ApoE3及ApoE4小鼠的遗传背景高度一致（转录组相似性＞99.8%）。与ApoE3小鼠相比，雌性ApoE4小鼠的癫痫样活动更显著、生存率更低，海马体剩余体积更小。三种基因型小鼠在缺氧缺血损伤后血糖水平均出现下降。ApoE4小鼠的酮体水平同样更低。在所有基因型的缺氧缺血组小鼠中，低血糖水平均与损伤后24小时海马体中促炎标志物及细胞死亡标志物水平升高相关，其中ApoE4小鼠的该关联更为显著；而当血糖水平较低且酮体水平偏高时，则与细胞死亡程度加剧（α-血影蛋白降解程度更高）密切相关。 结论：相较于ApoE3，人源化ApoE4会在新生小鼠缺氧缺血损伤后引发更严重的海马体损伤、细胞死亡及炎症反应，且该现象与低血糖及酮体利用不足相关。上述关联背后的具体机制仍有待进一步研究。