Neuroscience Program, University of Iowa Carver College of Medicine, Iowa City, IA, United States.
Department of Pediatrics, University of Iowa, Carver College of Medicine, Iowa City, IA, United States.
Alcohol. 2022 Jun;101:27-35. doi: 10.1016/j.alcohol.2022.02.005. Epub 2022 Apr 1.
Exposure to alcohol during pregnancy can kill developing fetal neurons and lead to fetal alcohol spectrum disorder (FASD) in the offspring. However, not all fetuses are equally vulnerable to alcohol toxicity. These differences in vulnerability among individuals are likely due, at least in part, to genetic differences. Some genes encode neuroprotective molecules that act through signaling pathways to protect neurons against alcohol's toxic effects. One signaling pathway that can protect cultured neurons against alcohol-induced cell death in vitro is the cAMP pathway. A goal of this study was to determine whether the cAMP pathway can exert a similar neuroprotective effect against alcohol in vivo. A key molecule within the cAMP pathway is cAMP response element binding protein (CREB). In this study, CREB was specifically disrupted in cerebellar Purkinje cells to study its role in protection of cerebellar neurons against alcohol toxicity.
Mice with Purkinje cell-specific knockout of CREB were generated with the Cre-lox system. A 2 × 2 design was used in which Cre-negative and Cre-positive mice received either 0.0 or 2.2 mg/g ethanol by intraperitoneal (i.p.) injection daily over postnatal day (PD) 4-9. Stereological cell counts of cerebellar Purkinje cells and granule cells were performed on PD 10. Motor function was assessed on PD 40 using the rotarod.
Purkinje cell-specific disruption of CREB alone (in the absence of alcohol) induced only a small reduction in Purkinje cell number. However, the loss of CREB function from Purkinje cells greatly increased the vulnerability of Purkinje cells to alcohol-induced cell death. While alcohol killed 20% of Purkinje cells in the Cre-negative (CREB-expressing) mice, alcohol killed 57% of Purkinje cells in the Cre-positive (CREB-nonexpressing) mice. This large loss of Purkinje cells did not lead to similar alcohol-induced losses of granule cells. In the absence of alcohol, lack of CREB function in Purkinje cells had no effect on rotarod performance. However, in the presence of alcohol, disruption of CREB in Purkinje cells substantially worsened rotarod performance.
Disruption of a single gene (CREB) in a single neuronal population (Purkinje cells) greatly increases the vulnerability of that cell population to alcohol-induced cell death and worsens alcohol-induced brain dysfunction. The results suggest that the cAMP pathway can protect cells in vivo against alcohol toxicity and underline the importance of genetics in determining the neuropathology and behavioral deficits of FASD.
怀孕期间接触酒精会杀死发育中的胎儿神经元，并导致后代出现胎儿酒精谱系障碍（FASD）。然而，并非所有胎儿对酒精毒性的敏感性都相同。个体之间这种敏感性的差异可能至少部分归因于遗传差异。一些基因编码神经保护分子，通过信号通路发挥作用，以保护神经元免受酒精的毒性影响。在体外，可保护培养神经元免受酒精诱导的细胞死亡的一种信号通路是 cAMP 通路。本研究的目的之一是确定 cAMP 通路是否能在体内对酒精发挥类似的神经保护作用。cAMP 通路中的关键分子是 cAMP 反应元件结合蛋白（CREB）。在这项研究中，使用 Cre-lox 系统特异性地破坏小脑浦肯野细胞中的 CREB，以研究其在保护小脑神经元免受酒精毒性方面的作用。
使用 Cre-lox 系统生成浦肯野细胞特异性 CREB 敲除的小鼠。采用 2×2 设计，在该设计中，Cre 阴性和 Cre 阳性小鼠在出生后第 4-9 天每天通过腹腔（i.p.）注射接受 0.0 或 2.2mg/g 乙醇。在第 10 天进行小脑浦肯野细胞和颗粒细胞的立体学细胞计数。在第 40 天使用转棒评估运动功能。
单独（没有酒精的情况下）破坏浦肯野细胞中的 CREB 仅导致浦肯野细胞数量略有减少。然而，从浦肯野细胞中丧失 CREB 功能大大增加了浦肯野细胞对酒精诱导的细胞死亡的敏感性。虽然酒精在 Cre 阴性（表达 CREB）小鼠中杀死了 20%的浦肯野细胞，但在 Cre 阳性（不表达 CREB）小鼠中，酒精杀死了 57%的浦肯野细胞。大量的浦肯野细胞损失并没有导致类似的酒精诱导的颗粒细胞损失。在没有酒精的情况下，浦肯野细胞中缺乏 CREB 功能对转棒表现没有影响。然而，在有酒精的情况下，破坏浦肯野细胞中的 CREB 大大恶化了转棒表现。
在单个神经元群体（浦肯野细胞）中破坏单个基因（CREB）大大增加了该细胞群体对酒精诱导的细胞死亡的敏感性，并恶化了酒精引起的脑功能障碍。结果表明，cAMP 通路可以保护体内细胞免受酒精毒性的影响，并强调了遗传在确定 FASD 的神经病理学和行为缺陷方面的重要性。
