Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. AIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

中文翻译：

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AIF3剪接开关触发神经变性

凋亡诱导因子 (AIF) 作为一种线粒体黄素蛋白，在线粒体生物能学中发挥着重要作用，对细胞存活至关重要，一旦从线粒体中释放出来并在缺血性中风或神经退行性疾病下转移到细胞核中，它也会介导不依赖于半胱天冬酶的细胞死亡疾病。尽管涉及 AIF 的选择性剪接调节，但尚不清楚在病理条件下会诱导哪种 AIF 剪接异构体以及它如何影响成人大脑中的线粒体功能和神经变性。通过多种方法确定脑中的 AIF 剪接诱导，包括 5' RACE、Sanger 测序、剪接特异性 PCR 测定和自下而上的蛋白质组学分析。AIF 剪接在线粒体和神经变性中的作用取决于其生化特性，细胞死亡分析、形态和功能改变以及动物行为。使用Cre-loxp重组或CRISPR/Cas9技术建立的三种动物模型，包括功能丧失模型、功能获得性AIF3敲入模型和条件诱导型AIF剪接模型，用于探索AIF剪接的潜在机制。诱发神经变性。我们鉴定了一种缺乏外显子 2 和 3 的自然剪接 AIF 异构体，称为 AIF3。AIF3 在生理条件下检测不到，但其在中风后小鼠和人类死后大脑中的表达增加。小鼠大脑中的 AIF3 剪接导致前脑区域扩大的心室和严重的神经变性。这些 AIF3 剪接小鼠在出生后 2-4 个月死亡。AIF3 剪接触发的神经变性涉及线粒体功能障碍和 AIF3 核易位。我们发现 AIF3 抑制 NADH 氧化酶活性、ATP 产生、耗氧量和线粒体生物发生。此外，AIF3 的表达显着增加染色质凝聚和核收缩，导致神经元细胞死亡。然而，丑角中单独的 AIF 损失或 AIF3 敲入小鼠中单独的 AIF3 增加并没有像在 AIF3 剪接小鼠中观察到的那样引起强烈的神经变性。我们将 AIF3 鉴定为疾病诱导型亚型，并建立了 AIF3 剪接小鼠模型。AIF3 剪接诱导的神经变性的分子机制涉及线粒体功能障碍和 AIF3 核易位，这是由 AIF 缺失和 AIF3 获得的协同效应引起的。