Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Mol Neurodegener. 2021 Apr 14;16(1):25. doi: 10.1186/s13024-021-00442-7.
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.
凋亡诱导因子（AIF）作为一种线粒体黄素蛋白，在细胞生存至关重要的线粒体生物能学中发挥着基本作用，并且在缺血性中风或神经退行性疾病下从线粒体释放并转移到核内后，介导 caspase 非依赖性细胞死亡。尽管已经暗示了 AIF 的可变剪接调节，但尚不清楚哪种 AIF 剪接异构体将在病理条件下被诱导，以及它如何影响成年大脑中的线粒体功能和神经退行性变。
通过包括 5'RACE、Sanger 测序、剪接特异性 PCR 测定和自上而下的蛋白质组学分析在内的多种方法确定脑内 AIF 剪接的诱导。通过其生化特性、细胞死亡分析、形态和功能改变以及动物行为来确定 AIF 剪接在线粒体和神经退行性变中的作用。应用三种动物模型，包括功能丧失性 harlequin 模型、功能获得性 AIF3 敲入模型和使用 Cre-loxp 重组或 CRISPR/Cas9 技术建立的条件诱导 AIF 剪接模型，探索 AIF 剪接诱导的神经退行性变的潜在机制。
我们鉴定出一种缺乏外显子 2 和 3 的天然剪接 AIF 异构体，命名为 AIF3。AIF3 在生理条件下无法检测到，但在中风后小鼠和人类死后的大脑中表达增加。在小鼠脑中剪接 AIF3 导致前脑区域的脑室扩大和严重的神经退行性变。这些 AIF3 剪接小鼠在出生后 2-4 个月死亡。AIF3 剪接触发的神经退行性变涉及线粒体功能障碍和 AIF3 核转位。我们表明，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 的表达显著增加染色质凝聚和核收缩，导致神经元细胞死亡。然而，harlequin 中的 AIF 缺失或 AIF3 敲入小鼠中的 AIF3 过表达本身并未引起如 AIF3 剪接小鼠中观察到的那样强烈的神经退行性变。
我们鉴定出 AIF3 是一种疾病诱导型异构体，并建立了 AIF3 剪接小鼠模型。AIF3 剪接诱导的神经退行性变的分子机制涉及线粒体功能障碍和 AIF3 核转位，这是由于 AIF 缺失和 AIF3 过表达的协同作用所致。我们的研究提供了一种理解 AIF3 剪接在大脑中作用的有价值的工具，并为预防/延缓神经退行性疾病的进展提供了潜在的治疗靶点。
