New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer’s disease
新型亲环蛋白 D 抑制剂可挽救阿尔茨海默氏病的线粒体和认知功能

Abstract 抽象的

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer’s disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer’s disease. However, there is currently no effective CypD inhibitor for Alzheimer’s disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood–brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer’s disease mitochondrial and glycolytic bioenergetics in Alzheimer’s disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer’s disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer’s disease mouse models.
线粒体功能障碍是阿尔茨海默病的早期病理特征，在阿尔茨海默病的发生和发展中发挥着至关重要的作用。拯救线粒体功能和认知的策略仍有待探索。亲环蛋白 D (CypD)，肽基脯氨酰异构酶 F (PPIase)，是打开线粒体膜通透性转换孔的关键成分，导致线粒体功能障碍和细胞死亡。通过抑制 CypD 活性来阻断膜通透性转换孔的开放是治疗阿尔茨海默病的一种有前景的方法。然而，目前尚无有效治疗阿尔茨海默病的 CypD 抑制剂，之前的候选药物毒性高、穿过血脑屏障的能力差、生物相容性差且选择性低。在此，我们报告了一类新型无毒且生物相容性 CypD 抑制剂依布硒啉 (ebselen)，它使用传统的 PPIase 测定法筛选约 2000 种 FDA 批准的药物库，并对 CypD-依布硒啉 (ebselen) 晶体结构进行晶体学分析（PDB 代码：8EJX） 。更重要的是，我们评估了 CypD 的遗传和药理学阻断对阿尔茨海默病衍生的线粒体 cybrid 细胞（一种离体人散发性阿尔茨海默病线粒体模型）中阿尔茨海默病线粒体和糖酵解生物能的影响，以及对突触功能、炎症反应和学习的影响。阿尔茨海默病小鼠模型的记忆。

Inhibition of CypD by ebselen protects against sporadic Alzheimer’s disease- and amyloid-β-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer’s disease mouse models, which is linked to CypD-related membrane permeability transition pore formation.
依布硒啉对 CypD 的抑制可防止偶发性阿尔茨海默病和淀粉样蛋白 β 诱导的线粒体和糖酵解扰动、突触和认知功能障碍，同时抑制阿尔茨海默病小鼠模型大脑中的神经炎症，这与 CypD 相关的膜通透性转变有关孔隙形成。

Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer’s disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.
因此，CypD 抑制剂有可能通过增强线粒体生物能并改善突触和认知功能来减缓神经退行性疾病（包括阿尔茨海默病）的进展。

Introduction 介绍

Alzheimer disease is the most common cause of dementia in the elderly, causing cognitive and memory impairment due to neuronal stress-induced cell death. Amyloid pathology and neurofibrillary tangles (NFT) with abnormally high phosphorylation of tau protein are two pathological features observed in Alzheimer’s disease-affected brains. However, the underlying mechanism and strategies to rescue this injury remain to be explored. Currently, there is no cure or effective treatment for Alzheimer’s disease. Most drugs approved for the treatment of Alzheimer’s disease provide only modest, temporary symptomatic improvement in cognitive function rather than slowing or reversing disease progression.¹ Recently approved amyloid-directed immunotherapies are able to reduce amyloid pathology, but these only modestly slow, rather than reverse, cognitive decline at an early stage of Alzheimer’s disease.^2,3 The safety and side effects of these amyloid-targeted drugs remain to be determined.^1,3 Clearly, there is an imperative need to develop more effective disease-modifying therapies for Alzheimer’s disease. Furthermore, delineating molecular and cellular mechanisms responsible for neurodegeneration and cognitive decline affords the opportunity to develop therapeutic targets to prevent or delay Alzheimer’s disease progression.
阿尔茨海默病是老年人痴呆的最常见原因，由于神经元应激诱导的细胞死亡而导致认知和记忆障碍。淀粉样蛋白病理学和 tau 蛋白异常高磷酸化的神经原纤维缠结 (NFT) 是在阿尔茨海默氏病影响的大脑中观察到的两种病理特征。然而，挽救这种损伤的潜在机制和策略仍有待探索。目前，阿尔茨海默病尚无治愈或有效的治疗方法。大多数被批准用于治疗阿尔茨海默病的药物只能对认知功能提供适度的、暂时的症状改善，而不是减缓或逆转疾病的进展。 ¹ 最近批准的淀粉样蛋白定向免疫疗法能够减少淀粉样蛋白病理，但这些只能适度减缓而不是逆转阿尔茨海默病早期认知能力的下降。 ^2,3 这些淀粉样蛋白靶向药物的安全性和副作用仍有待确定。 ^1,3 显然，迫切需要开发针对阿尔茨海默病的更有效的疾病缓解疗法。此外，描述导致神经变性和认知能力下降的分子和细胞机制为开发治疗靶点以预防或延缓阿尔茨海默病的进展提供了机会。

The causes of Alzheimer’s disease likely include a combination of age-related changes in the brain along with a variety of factors that increase the risk of Alzheimer’s disease. Emerging studies shed light on the contribution of mitochondria to the onset and advancement of neurodegeneration and cognitive decline. Studies from a large number of independent groups provide substantial evidence of Alzheimer’s disease- or amyloid-β (Aβ)/tau-related mitochondrial dysfunction in the brains of Alzheimer’s disease patients, Alzheimer’s disease mouse models^4-33 and Alzheimer’s disease-derived mitochondria in cybrid cells.^13,16,34-46 This includes decreased activity of key enzymes involved in mitochondrial respiratory chain function and abnormalities in mitochondrial dynamics and energy metabolism.^47-51 Although the advanced Alzheimer’s disease brain is characterized by Aβ deposition and NFT formation, synaptic mitochondrial damage may precede memory loss and these hallmark lesions in Alzheimer’s disease patients.^45,52 This may explain why anti-Aβ therapy has limited benefit in late-onset, sporadic Alzheimer’s disease. Strategies to rescue or enhance mitochondrial and cognitive function in Alzheimer’s disease patients remain to be discovered.
阿尔茨海默病的原因可能包括与年龄相关的大脑变化以及各种增加阿尔茨海默病风险的因素的结合。新兴研究揭示了线粒体对神经退行性变和认知能力下降的发生和进展的贡献。来自大量独立团体的研究提供了大量证据，证明阿尔茨海默病患者、阿尔茨海默病小鼠模型 ^4-33 和阿尔茨海默病的大脑中存在阿尔茨海默病或淀粉样蛋白-β (Aβ)/tau 相关的线粒体功能障碍。 -细胞杂种细胞中衍生的线粒体。 ^13,16,34-46 这包括参与线粒体呼吸链功能的关键酶活性降低以及线粒体动力学和能量代谢异常。 ^47-51 虽然晚期阿尔茨海默病大脑的特点是 Aβ 沉积和 NFT 形成，但突触线粒体损伤可能先于记忆丧失和阿尔茨海默病患者的这些标志性病变。 ^45,52 这或许可以解释为什么抗 Aβ 疗法对迟发性、散发性阿尔茨海默病的疗效有限。拯救或增强阿尔茨海默病患者线粒体和认知功能的策略仍有待发现。

Cyclophilin D (CypD), a peptidyl prolyl cis-trans isomerase F (PPIase) located in the mitochondrial matrix, is a crucial component of mitochondrial permeability transition pore (mPTP) formation.^44,53,54 MPTP opening results in osmotic swelling and consequent dissipation of mitochondrial potential, reduced mitochondrial calcium retention capacity, decreased membrane potential (ΔΨm) and increased reactive oxygen species (ROS) production, all of which ultimately lead to cell death.^55,56 Accordingly, genetic deficiency of CypD results in resistance to mPTP-related mitochondrial dysfunction and persistent life-long protection against Aβ toxicity and cognitive impairment in Alzheimer’s disease mouse models.^44,57 However, the direct link between CypD and mitochondrial defects found in sporadic Alzheimer’s disease mitochondria remains elusive. In addition to Alzheimer’s disease models, the protective effects of CypD loss are well documented in the disease phenotypes of animal models of ischaemia/reperfusion injury, trauma and multiple sclerosis.^44,52,57-61 Inhibition of CypD by cyclosporin A (CsA), a specific CypD inhibitor that binds to CypD and blocks its PPIase activity, prevents Aβ- and oxidative stress-induced mitochondrial dysfunction and synaptic degeneration.^{44,46,56,57,61} Unfortunately, CsA lacks clinical significance in disorders associated with the CNS because of its immunosuppressive effect, severe side effects, and inability to cross the blood­–brain barrier (BBB). Sanglifehrin A and antamanide were recently developed as mPTP-inhibiting drugs.^62,63 However, the side effects including nephrotoxicity, neurotoxicity, and hepatotoxicity as well as poor BBB permeability remain unclear. The clear need for improved CypD inhibitors led us to screen a library of FDA approved drugs commonly used in repurposing studies, leading us to identify ebselen as a novel CypD inhibitor.
亲环蛋白 D (CypD) 是一种位于线粒体基质中的肽基脯氨酰顺反异构酶 F (PPIase)，是线粒体通透性转换孔 (mPTP) 形成的重要组成部分。 ^44,53,54 MPTP 打开导致渗透性膨胀，从而导致线粒体电位耗散、线粒体钙保留能力降低、膜电位 (ΔΨm) 降低和活性氧 (ROS) 产生增加，所有这些最终导致细胞死亡。 ^55,56 因此，CypD 的遗传缺陷导致对 mPTP 相关线粒体功能障碍的抵抗，并在阿尔茨海默病小鼠模型中持续终生抵抗 Aβ 毒性和认知障碍。 ^44,57 然而，CypD 与散发性阿尔茨海默病线粒体中发现的线粒体缺陷之间的直接联系仍然难以捉摸。除了阿尔茨海默病模型外，CypD 缺失的保护作用在缺血/再灌注损伤、创伤和多发性硬化症动物模型的疾病表型中也得到了充分证明。 ^44,52,57-61 环孢菌素 A (CsA) 是一种特异性 CypD 抑制剂，可与 CypD 结合并阻断其 PPIase 活性，从而抑制 CypD，从而防止 Aβ 和氧化应激诱导的线粒体功能障碍和突触变性。 ^{44,46,56,57,61} 不幸的是，CsA 由于其免疫抑制作用、严重的副作用以及无法穿过血脑屏障 (BBB)，因此在中枢神经系统相关疾病中缺乏临床意义。 Sanglifehrin A 和antamanide 最近被开发为mPTP 抑制药物。 ^62,63 然而，其肾毒性、神经毒性、肝毒性以及血脑屏障通透性差等副作用仍不清楚。 对改进 CypD 抑制剂的明确需求促使我们筛选了 FDA 批准的常用于再利用研究的药物库，使我们确定依布硒啉是一种新型 CypD 抑制剂。

Based on preclinical studies and phase I–III clinical trials, ebselen is a bioavailable, BBB permeant, safe and cell protective compound.^64,65 The protection of ebselen against oxidative stress-induced cellular dysfunction has been reported by other independent groups,^66-74 with the most recent studies performed in Alzheimer’s disease-related mouse models.^75-77 However, the underlying mechanism of ebselen on Alzheimer’s disease-related mitochondrial dysfunction has not yet been explored. Thus, it is important and rationale to elucidate mechanisms underlying the protective effects of ebselen linked to inhibiting CypD-mediated mitochondrial mPTP relevant to Alzheimer’s disease- and Aβ-mediated mitochondrial dysfunction. This will greatly facilitate the development of Alzheimer’s disease-modified therapeutic targets to improve mitochondrial and cognitive function.
根据临床前研究和 I-III 期临床试验，依布硒啉是一种生物可利用、可渗透 BBB、安全且具有细胞保护作用的化合物。 ^64,65 其他独立研究小组也报道了依布硒啉对氧化应激诱导的细胞功能障碍的保护作用， ^66-74 最近的研究是在阿尔茨海默病相关的小鼠模型中进行的。 ^75-77 然而，依布硒啉对阿尔茨海默病相关线粒体功能障碍的潜在机制尚未被探索。因此，阐明依布硒啉保护作用的机制是重要且合理的，该作用与抑制与阿尔茨海默病和 Aβ 介导的线粒体功能障碍相关的 CypD 介导的线粒体 mPTP 相关。这将极大地促进阿尔茨海默病改良治疗靶点的开发，以改善线粒体和认知功能。

To explore the consequences and underlying mechanisms associated with Alzheimer’s disease-specific mitochondrial defects, we recently generated a transmitochondrial cytoplasmic hybrid (cybrid) neuronal cell line, consisting of a unique mitochondrial DNA (mtDNA)-depleted neuronal cell line incorporated with platelet mitochondria derived from sporadic Alzheimer’s disease patients to model human Alzheimer’s disease-relevant abnormal mitochondrial and glycolytic bioenergetics/functions. These human Alzheimer’s disease cybrids recapitulate many pathological and mitochondrial changes seen in Alzheimer’s disease brains including mitochondrial and synaptic alterations and elevated Aβ/abnormal tau protein.^{34,40,43,78-81} Alzheimer’s disease platelet mitochondria exhibit decreased complex activity associated with ROS overproduction, reduced maximal capacity of the electron transport system and reduced respiration rates.^82-84 Furthermore, CypD levels were significantly increased alongside mitochondrial failure in Alzheimer’s disease cybrids. Thus, it is essential to use this unique Alzheimer’s disease cellular model to investigate the causes and consequences of Alzheimer’s disease-related mitochondrial dysfunction and to validate the biological efficacy of small molecules, such as CypD blockers.
为了探索与阿尔茨海默病特异性线粒体缺陷相关的后果和潜在机制，我们最近生成了一种传递软骨细胞质杂合（cybrid）神经元细胞系，该细胞系由一种独特的线粒体DNA（mtDNA）耗尽的神经元细胞系与源自血小板线粒体的细胞系组成。散发性阿尔茨海默病患者模拟人类阿尔茨海默病相关的异常线粒体和糖酵解生物能/功能。这些人类阿尔茨海默病细胞重现了阿尔茨海默病大脑中观察到的许多病理和线粒体变化，包括线粒体和突触改变以及 Aβ/异常 tau 蛋白升高。 ^{34,40,43,78-81} 阿尔茨海默病血小板线粒体表现出与 ROS 过量产生相关的复杂活性降低、电子传输系统最大容量降低和呼吸速率降低。 ^82-84 此外，在阿尔茨海默氏病细胞杂种中，CypD 水平随着线粒体衰竭而显着增加。因此，有必要利用这种独特的阿尔茨海默病细胞模型来研究阿尔茨海默病相关线粒体功能障碍的原因和后果，并验证小分子（如 CypD 阻滞剂）的生物学功效。

Materials and methods 材料和方法

Human subjects and creation of cybrid cell lines
人类受试者和细胞系的创建

Individuals for this study were recruited from the University of Kansas Alzheimer’s Disease Center. Alzheimer’s disease subjects met the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer disease and Related Disorders Association.⁸⁵ Non-Alzheimer’s disease subjects were cognitively normal and age-matched to Alzheimer’s disease subjects (Supplementary material).
这项研究的受试者是从堪萨斯大学阿尔茨海默病中心招募的。阿尔茨海默病受试者符合美国国家神经和交流障碍与中风研究所以及阿尔茨海默病和相关疾病协会的标准。 ⁸⁵ 非阿尔茨海默病受试者认知正常，并且与阿尔茨海默病受试者年龄匹配（补充材料）。

Cybrid cell lines were created on the human neuroblastoma cell (SH-SY5Y) nuclear background (by the KU ADC Mitochondrial Genomics and Metabolism Core).⁸⁶ The Rho0 cells (cells lacking mtDNA) were produced by culturing SH-SY5Y cells in the presence of ethidium bromide. Rho0 cells were then fused with the platelet cytoplasm and repopulated with mitochondria containing mtDNA from patients or controls, as previously described.⁸⁷ Quantitative real-time PCR showed that intact mtDNA copies were present in all cybrids with no detectable large-scale deletion after many passages of cell proliferation.⁵⁰ Five non-Alzheimer’s disease and six Alzheimer’s disease cybrid cell lines from five and six human subjects were used in this study (Supplementary Table 1).
Cybrid 细胞系是在人神经母细胞瘤细胞 (SH-SY5Y) 核背景上创建的（由 KU ADC 线粒体基因组学和代谢核心）。 ⁸⁶ Rho0细胞（缺乏mtDNA的细胞）是通过在溴化乙锭存在下培养SH-SY5Y细胞而产生的。然后将 Rho0 细胞与血小板细胞质融合，并用含有来自患者或对照的 mtDNA 的线粒体进行重新填充，如前所述。 ⁸⁷ 实时定量PCR显示，在细胞增殖多次传代后，所有胞质杂种中均存在完整的mtDNA拷贝，未检测到大规模缺失。 ⁵⁰ 本研究使用了来自 5 名和 6 名人类受试者的 5 种非阿尔茨海默病和 6 种阿尔茨海默病 cybrid 细胞系（补充表 1）。

Mice 老鼠

All studies with mice were performed in accordance to the National Institutes of Health guidelines for animal care with the approval of the Institutional Animal Care and Use Committee of the University of Kansas-Lawrence and Columbia University. Transgenic mice overexpressing a mutant human form of amyloid precursor protein (APP) that encodes hAPP695, hAPP751 and hAPP770 bearing mutations linked to familial Alzheimer’s disease [APPV717F (Indiana), KM670/671NL(Swedish), J-20 line and 5xFAD mice were obtained from Jackson Laboratory].⁸⁸ Offspring of transgenic (Tg) mice were identified by PCR using primers for each specific transgene, as previously described.⁵³ Both male and female genders were used in the study.
所有小鼠研究均按照美国国立卫生研究院动物护理指南进行，并得到堪萨斯劳伦斯大学和哥伦比亚大学动物护理和使用机构委员会的批准。转基因小鼠过度表达突变型人类淀粉样前体蛋白 (APP)，编码 hAPP695、hAPP751 和 hAPP770，这些小鼠携带与家族性阿尔茨海默病相关的突变 [APPV717F（印第安纳州）、KM670/671NL（瑞典）、J-20 系和 5xFAD 小鼠来自杰克逊实验室]。 ⁸⁸ 如前所述，使用每个特定转基因的引物通过 PCR 鉴定转基因 (Tg) 小鼠的后代。 ⁵³ 研究中使用了男性和女性。

Cybrid cell culture for Seahorse assays
用于 Seahorse 检测的 Cybrid 细胞培养

Non-Alzheimer’s disease and Alzheimer’s disease cybrid cells were cultured (20,000 cells per well) on XF96 Cell Culture Microplate (Cat. No. 102601-100) using Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). After 24 h of seeding, growing media was replaced with serum-free DMEM media and/or cells were incubated with vehicle or different compounds (CsA, mitoTEMPO, and ebselen) for 24 h under cell growth conditions (5% CO₂ and 37°C). Cells were washed and incubated with assay media (Seahorse DMEM pH 7.4 containing 10 mM glucose and 2 mM glutamine) for 1 h at 37°C (non-CO₂ incubator) before performing various Seahorse assays. Seahorse XF cartridges were activated with XFe96 calibrant (Cat. No. 100840-000) and hydrated overnight.
使用含有 10% 胎牛血清 (FBS) 和 1 ％青霉素-链霉素(PS)。接种 24 小时后，将生长培养基更换为无血清 DMEM 培养基和/或将细胞与载体或不同化合物（CsA、mitoTEMPO 和 ebselen）在细胞生长条件下（5% CO ₂ 培养箱）下孵育 1 小时。 Seahorse XF 柱用 XFe96 校准剂（货号 100840-000）激活并水合过夜。

Measurement of mitochondrial respiration and glycolytic function and rate of ATP production
线粒体呼吸和糖酵解功能以及 ATP 生成率的测量

All reagents and accessories were obtained from Agilent Technologies, including Seahorse XF Cell Mito Stress Assay Kit (Cat. No. 103015-100), Seahorse XF Glycolytic Rate Assay Kit (Cat. No. 103344-100) and Seahorse XF Real-time ATP Rate Assay Kit (Cat. No. 103592-100). The real time mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe96 Extracellular Flux Analyzer following the manufacturer’s instructions. OCR and ECAR were measured upon the sequential addition of oligomycin (2.5 µM), FCCP (2.0 µM) and rotenone/antimycin A (0.5 µM) to the experimental cells. To assess glycolytic function, OCR, ECAR and proton efflux rate (PER) were measured using the Seahorse XFe96 Extracellular Flux Analyzer, following the manufacturer’s instructions. OCR, ECAR and PER were measured following the sequential addition of rotenone/antimycin A (0.5 µM) and 2-deoxy-D-glucose (50 mM) to the experimental cells. The rate of mitochondrial and glycolytic ATP production was measured using the Seahorse XFe96 Extracellular Flux Analyzer, following the manufacturer’s instructions. ATP production was measured upon sequential addition of oligomycin (2.5 µM) and rotenone/antimycin A (0.5 µM) to the experimental cells. Data were normalized by final protein concentration on each well of Seahorse plate after experiments. The raw data were analysed using Seahorse Analyzer and GraphPad Prism 9 software.
所有试剂和配件均来自安捷伦科技公司，包括 Seahorse XF 细胞线粒体应激测定试剂盒（货号 103015-100）、Seahorse XF 糖酵解速率测定试剂盒（货号 103344-100）和 Seahorse XF 实时 ATP速率测定试剂盒（货号 103592-100）。使用 Seahorse XFe96 细胞外通量分析仪按照制造商的说明测量实时线粒体耗氧率 (OCR) 和细胞外酸化率 (ECAR)。 OCR 和 ECAR 是在向实验细胞中依次添加寡霉素 (2.5 μM)、FCCP (2.0 μM) 和鱼藤酮/抗霉素 A (0.5 μM) 时测量的。为了评估糖酵解功能，按照制造商的说明，使用 Seahorse XFe96 细胞外通量分析仪测量 OCR、ECAR 和质子流出率 (PER)。在向实验细胞中连续添加鱼藤酮/抗霉素 A (0.5 μM) 和 2-脱氧-D-葡萄糖 (50 mM) 后，测量 OCR、ECAR 和 PER。使用 Seahorse XFe96 细胞外通量分析仪按照制造商的说明测量线粒体和糖酵解 ATP 的产生速率。向实验细胞中连续添加寡霉素 (2.5μM) 和鱼藤酮/抗霉素 A (0.5μM) 后测量 ATP 产量。实验后，通过海马板每孔的最终蛋白质浓度对数据进行标准化。使用 Seahorse Analyzer 和 GraphPad Prism 9 软件对原始数据进行分析。

Pharmacological treatment
药物治疗

For mouse treatment, ebselen (Cat. No. 70530; Cayman chemical) was dissolved in 0.4% dimethylsulphoxide (DMSO) followed by addition of 40% PEG and 59.6% sterile saline (0.9%), and then passed through a 0.22 µm filter to sterilize the ebselen solution prior to use. mAPP mice (11–12 months of age) or 5xFAD mice (6 months of age) with their littermate nonTg controls were subjected to intraperitoneal injection (i.p.) at a dosage of 2.5 mg/kg/day or sterile vehicle with the same volume for 8 weeks. To test the inhibitory effect of ebselen on CypD activity in mouse brains, mice were treated with ebselen (2.5 mg/kg, i.p. injection) for 4–6 weeks.
对于小鼠治疗，将依布硒啉（货号 70530；Cayman Chemical）溶解在 0.4% 二甲基亚砜 (DMSO) 中，然后添加 40% PEG 和 59.6% 无菌盐水 (0.9%)，然后通过 0.22 μm 过滤器使用前对依布硒啉溶液进行灭菌。 mAPP 小鼠（11-12 月龄）或 5xFAD 小鼠（6 月龄）及其同窝非 Tg 对照小鼠以 2.5 mg/kg/天的剂量或相同体积的无菌载体进行腹膜内注射 (i.p.)。 8周。为了测试依布硒啉对小鼠大脑 CypD 活性的抑制作用，用依布硒啉（2.5 mg/kg，腹腔注射）治疗小鼠 4-6 周。

Additional methods 附加方法

Additional methods are available in the online Supplementary material, ‘Methods’ section and include the following: measurement of enzyme activities associated with respiratory chain complexes and ATP levels in brain of Alzheimer’s disease mice; mitochondrial membrane potential analysis; mitochondrial permeability transition pore opening; reactive oxygen species measurement; PPIF siRNA knockdown and mitochondrial function; cyclophilin D overexpression; quantitative real-time PCR analysis; immunoblot analysis; immunofluorescent staining of cybrid cells; high throughput screen of the FDA drug library; long-term potentiation recording; and behavioural tests.
其他方法可在在线补充材料的“方法”部分中找到，包括以下内容：测量阿尔茨海默病小鼠大脑中与呼吸链复合物和 ATP 水平相关的酶活性；线粒体膜电位分析；线粒体通透性转变孔开放；活性氧测量； PPIF siRNA 敲低和线粒体功能；亲环蛋白 D 过度表达；实时定量PCR分析；免疫印迹分析； cybrid细胞的免疫荧光染色； FDA药物库的高通量筛选；长时程增强记录；和行为测试。

Statistical analysis 统计分析

All data are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined by unpaired t-test and one-way ANOVA according to the number of independent variables using commercially available GraphPad Prism 9 software. Fisher multiple comparisons test was performed for post hoc comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
所有数据均以平均值±平均值标准误差 (SEM) 的形式呈现。使用市售的 GraphPad Prism 9 软件，根据自变量的数量，通过不配对 t 检验和单向方差分析确定统计显着性。进行费舍尔多重比较检验进行事后比较。 *P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。

Results 结果

Increased expression of CypD and impaired mPTP in Alzheimer’s disease cybrid cells
阿尔茨海默病 cybrid 细胞中 CypD 表达增加和 mPTP 受损

As the first step towards understanding the role of CypD in sporadic Alzheimer’s disease-derived mitochondria in neuronal cybrid cells, we investigated the expression levels of CypD in Alzheimer’s disease cybrid cells containing sporadic Alzheimer’s disease mitochondria. CypD transcript and protein expression levels were significantly increased in Alzheimer’s disease cybrid cells compared to non-Alzheimer’s disease cybrid cells by qPCR, immunoblotting and immunostaining (Fig. 1A–D). These data suggest that increased expression of CypD is associated with mitochondrial defects in human Alzheimer’s disease.
作为了解 CypD 在神经元 cybrid 细胞中散发性阿尔茨海默病衍生线粒体中作用的第一步，我们研究了含有散发性阿尔茨海默病线粒体的阿尔茨海默病 cybrid 细胞中 CypD 的表达水平。通过 qPCR、免疫印迹和免疫染色显示，与非阿尔茨海默病 cybrid 细胞相比，阿尔茨海默病 cybrid 细胞中 CypD 转录物和蛋白表达水平显着增加（图 1A-D）。这些数据表明 CypD 表达的增加与人类阿尔茨海默氏病的线粒体缺陷有关。

In view of the role of CypD in mPTP formation and increased expression of CypD in Alzheimer’s disease-associated mitochondria, we measured mPTP opening in non-Alzheimer’s disease and Alzheimer’s disease cybrids under basal conditions using the CoCl₂-calcein fluorescence-quenching assay. Cells were incubated with calcein-AM, a membrane permeable fluorophore that freely diffuses in all subcellular compartments, including mitochondria, in the presence of divalent cobalt cations (Co²⁺), which is capable of quenching calcein fluorescence in all subcellular compartments, except the mitochondrial matrix, as the inner mitochondrial membrane is the only intracellular membrane that is impermeable to Co²⁺.⁸⁹ When mPTP is open, cobalt is able to enter mitochondria and quench mitochondrial calcein fluorescence.⁹⁰ In the presence of Co²⁺, the calcein fluorescent signal was significantly reduced in Alzheimer’s disease cybrid cells compared to non-Alzheimer’s disease cybrid cells (Fig. 1E and F). In the absence of Co²⁺, no significant difference in the calcein fluorescence intensity was found between Alzheimer’s disease and non-Alzheimer’s disease cybrid cells (Supplementary Fig. 1). These results suggest that mPTP is formed in Alzheimer’s disease-derived mitochondria. Accordingly, Alzheimer’s disease cybrid cells exhibited significantly reduced mitochondrial respiration, including basal and maximal oxygen consumption, ATP-coupled mitochondrial respiration and mitochondrial ATP levels (Fig. 1G–K).
鉴于CypD在mPTP形成中的作用以及阿尔茨海默病相关线粒体中CypD表达增加，我们使用CoCl ₂ -钙黄绿素在基础条件下测量了非阿尔茨海默病和阿尔茨海默病细胞杂种中mPTP的开放情况荧光猝灭测定。将细胞与钙黄绿素-AM 一起孵育，钙黄绿素-AM 是一种膜渗透性荧光团，在二价钴阳离子 (Co ²⁺ ) 存在的情况下，可在包括线粒体在内的所有亚细胞区室中自由扩散，能够猝灭所有亚细胞区室中的钙黄绿素荧光。亚细胞区室，除了线粒体基质之外，因为线粒体内膜是唯一不透 Co ²⁺ 的细胞内膜。 ⁸⁹ 当 mPTP 打开时，钴能够进入线粒体并猝灭线粒体钙黄绿素荧光。 ⁹⁰ 在 Co ²⁺ 存在的情况下，与非阿尔茨海默病 cybrid 细胞相比，阿尔茨海默病 cybrid 细胞中钙黄绿素荧光信号显着降低（图 1E 和 F）。在没有Co ²⁺ 的情况下，阿尔茨海默病和非阿尔茨海默病cybrid细胞之间的钙黄绿素荧光强度没有显着差异（补充图1）。这些结果表明 mPTP 是在阿尔茨海默病衍生的线粒体中形成的。因此，阿尔茨海默病细胞杂种细胞表现出显着降低的线粒体呼吸，包括基础耗氧量和最大耗氧量、ATP耦合线粒体呼吸和线粒体ATP水平（图1G-K）。

Effect of Alzheimer’s disease mitochondria on glycolytic function in Alzheimer’s disease cybrid neurons
阿尔茨海默病线粒体对阿尔茨海默病细胞杂种神经元糖酵解功能的影响

Mitochondria-related energy exhaustion may precede glycolysis-related hypometabolism in early neurodegeneration in Alzheimer’s disease.⁹¹ Metabolism of various fuel molecules generate and utilize energy through glycolysis, mitochondrial respiration and the pentose phosphate pathway (PPP), which together produce ATP and other essential metabolites. Recent studies suggest that glycolytic metabolism may play a critical role in Alzheimer’s disease development. Reduced glycolytic flux has been shown to correlate with the severity of amyloid and tau pathology in preclinical and clinical Alzheimer’s disease patients.⁹² This prompted us to evaluate the involvement of CypD in glycolytic energy metabolism in relation to mitochondrial bioenergetic failure in Alzheimer’s disease. To determine the glycolytic PER of cells, we directly measured real-time OCR together with ECAR stimulated with 2-DG. It is clear that Alzheimer’s disease cybrid cells not only displayed lower OCR, but also had significantly lower rates of glycolytic flux, including ECAR, PER, basal and maximal glycolytic capacity and glycolytic ATP production compared to non-Alzheimer’s disease cells (Fig. 2A–F). These results suggest that both mitochondrial respiration and glycolytic bioenergy metabolisms are perturbed in Alzheimer’s disease-derived mitochondria.
在阿尔茨海默病的早期神经变性中，线粒体相关的能量耗尽可能先于糖酵解相关的代谢低下。 ⁹¹ 各种燃料分子的代谢通过糖酵解、线粒体呼吸和磷酸戊糖途径（PPP）产生和利用能量，这些途径共同产生 ATP 和其他必需代谢物。最近的研究表明，糖酵解代谢可能在阿尔茨海默病的发展中发挥关键作用。在临床前和临床阿尔茨海默病患者中，糖酵解通量减少已被证明与淀粉样蛋白和 tau 蛋白病理的严重程度相关。 ⁹² 这促使我们评估 CypD 在糖酵解能量代谢中的参与与阿尔茨海默病中线粒体生物能衰竭的关系。为了确定细胞的糖酵解 PER，我们直接测量实时 OCR 以及 2-DG 刺激的 ECAR。很明显，与非阿尔茨海默病细胞相比，阿尔茨海默病细胞不仅表现出较低的 OCR，而且糖酵解通量率也显着降低，包括 ECAR、PER、基础和最大糖酵解能力以及糖酵解 ATP 产量（图 2A- F）。这些结果表明，在阿尔茨海默病衍生的线粒体中，线粒体呼吸和糖酵解生物能代谢均受到干扰。

Although we have demonstrated a protective effect of loss or inhibition of CypD on Aβ-mediated mitochondrial dysfunction in an Aβ-producing mouse model of Alzheimer’s disease,⁴⁴ little is known about the involvement of CypD in sporadic human Alzheimer’s disease mitochondrial respiration and glycolysis. To this end, we examined the effect of blocking CypD on Alzheimer’s disease-mediated changes in mitochondrial respiration and glycolysis through pharmacological CypD inhibition and genetic CypD knockdown. First, Alzheimer’s disease cybrid cells were treated with CsA, a well known CypD-specific inhibitor for 24 h, and then mitochondrial respiration was assessed. Basal and maximal OCR and ATP-related respiration were significantly increased in CsA-treated Alzheimer’s disease cybrid cells compared to vehicle treatment (Supplementary Fig. 2A–D). Second, Alzheimer’s disease cybrid cells were treated with CypD siRNA targeting the Ppif gene to determine the effect of CypD knockdown on mitochondrial respiration. CypD expression levels were reduced by ∼60% in cells treated with Ppif-siRNA compared to control siRNA-treated cells (Supplementary Fig. 2E). The effectiveness of Ppif-siRNA treatment was also confirmed through immunostaining with CypD antibody (Supplementary Fig. 2F and G). OCR and ATP levels in Alzheimer’s disease cybrid cells treated with Ppif-siRNA were significantly higher than those in control siRNA-treated cells (Supplementary Fig. 2H–K). Similarly, adding CsA (Supplementary Fig. 3A–E) or silencing CypD gene expression with Ppif-siRNA (Supplementary Fig. 3F–J) significantly elevated basal and glycolytic capacity in Alzheimer’s disease cybrids. Thus, inhibition or blockade of CypD rescues Alzheimer’s disease mitochondrial and glycolytic bioenergetic defects.
尽管我们已经在产生 Aβ 的阿尔茨海默病小鼠模型中证明了 CypD 的缺失或抑制对 Aβ 介导的线粒体功能障碍具有保护作用，但关于 CypD 在散发性人类阿尔茨海默病中的作用仍知之甚少。线粒体呼吸和糖酵解。为此，我们通过药理学 CypD 抑制和基因 CypD 敲低，研究了阻断 CypD 对阿尔茨海默病介导的线粒体呼吸和糖酵解变化的影响。首先，用 CsA（一种众所周知的 CypD 特异性抑制剂）处理阿尔茨海默病 cybrid 细胞 24 小时，然后评估线粒体呼吸。与媒介物处理相比，CsA 处理的阿尔茨海默病 cybrid 细胞的基础和最大 OCR 以及 ATP 相关呼吸显着增加（补充图 2A-D）。其次，用针对 Ppif 基因的 CypD siRNA 处理阿尔茨海默病 cybrid 细胞，以确定 CypD 敲低对线粒体呼吸的影响。与对照 siRNA 处理的细胞相比，用 Ppif-siRNA 处理的细胞中 CypD 表达水平降低了约 60%（补充图 2E）。 Ppif-siRNA 治疗的有效性也通过 CypD 抗体的免疫染色得到了证实（补充图 2F 和 G）。用 Ppif-siRNA 处理的阿尔茨海默病 cybrid 细胞中的 OCR 和 ATP 水平显着高于对照 siRNA 处理的细胞（补充图 2H-K）。同样，添加CsA（补充图3A-E）或用Ppif-siRNA沉默CypD基因表达（补充图3F-J）显着提高了阿尔茨海默病细胞的基础能力和糖酵解能力。 因此，抑制或阻断 CypD 可挽救阿尔茨海默病线粒体和糖酵解生物能缺陷。

Effect of mitochondrial ROS on mitochondrial respiratory and glycolytic function in Alzheimer’s disease cybrids
线粒体ROS对阿尔茨海默病细胞线粒体呼吸和糖酵解功能的影响

Given that increased mitochondrial ROS is an important contributor to aberrant mitochondrial structure and function^44,46,61 and that mPTP opening can be affected by elevated oxidative stress,⁴⁴ we next determined the link between mitochondrial ROS and Alzheimer’s disease mitochondrial and glycolytic perturbation. As shown in Fig. 3, scavenging mitochondrial ROS by applying a mitochondria-targeted antioxidant mitoTEMPO significantly increased basal and maximal OCR and ATP-coupled respiration (Fig. 3A–D) and enhanced glycolytic flux rate (ECAR and PER) and basal and maximal PER in Alzheimer’s disease cybrids (Fig. 3E–I). Thus, suppression of mitochondrial ROS could rescue mitochondrial respiration and glycolytic capacity in human Alzheimer’s disease-related mitochondrial injury.
鉴于增加的线粒体 ROS 是导致线粒体结构和功能异常的重要因素 ^44,46,61 ，并且 mPTP 开放可能受到氧化应激升高的影响， ⁴⁴ 我们接下来确定了线粒体 ROS 与线粒体功能之间的联系。阿尔茨海默病线粒体和糖酵解扰动。如图 3 所示，通过应用线粒体靶向抗氧化剂 mitoTEMPO 清除线粒体 ROS，可显着增加基础和最大 OCR 以及 ATP 耦合呼吸（图 3A-D），并增强糖酵解通量率（ECAR 和 PER）以及基础和最大 OCR。阿尔茨海默病细胞杂种中的 PER（图 3E-I）。因此，抑制线粒体ROS可以挽救人类阿尔茨海默病相关线粒体损伤中的线粒体呼吸和糖酵解能力。

Identification of new cyclophilin D inhibitors
新型亲环蛋白 D 抑制剂的鉴定

Known CypD inhibitors, including CsA, one of the most potent inhibitors for CypD, have one or both of the following disadvantages: poor BBB permeability and high toxicity. Clearly, it is essential to identify novel small molecules that can specifically bind to CypD and inhibit CypD-dependent mitochondrial perturbation. To this end, we screened a library of FDA approved drugs to identify potential new CypD inhibitors for their translational implication.
已知的CypD抑制剂，包括CsA（CypD最有效的抑制剂之一），具有以下一个或两个缺点：血脑屏障通透性差和毒性高。显然，有必要识别能够特异性结合 CypD 并抑制 CypD 依赖性线粒体扰动的新型小分子。为此，我们筛选了 FDA 批准的药物库，以确定潜在的新型 CypD 抑制剂的转化意义。

CypD activity was measured using a previously published protocol based on an assay designed by Fischer et al.⁹³ and later adapted for high throughput screens by Mori et al.⁹⁴ Detailed methods and data for assay optimization and screening can be found in the Supplementary material. Briefly, a fluorogenic peptide is trapped in the cis-proline conformation by resuspension in a non-aqueous solvent. Once diluted into the aqueous CypD solution, the substrate is isomerized to the trans-proline form, at which point it becomes a substrate for chymotrypsin and the AMC fluorophore is released. The reaction is optimized to run fast (i.e. ∼95% substrate conversion in 20 s) so that the CypD activity outpaces the spontaneous isomerization of the peptide. This necessitates the use of a reader like the FDSS7000 (Hamamatsu Photonics) that can record signals from all plate wells simultaneously on a sub-second timescale.
CypD 活性使用先前发布的基于 Fischer 等人设计的测定法的方案进行测量。 ⁹³ ，后来由 Mori 等人改编为高通量筛选。 ⁹⁴ 测定优化和筛选的详细方法和数据可以在补充材料中找到。简而言之，通过重悬于非水溶剂中，荧光肽被捕获在顺式脯氨酸构象中。一旦稀释到 CypD 水溶液中，底物就会异构化为反式脯氨酸形式，此时它成为胰凝乳蛋白酶的底物，并释放 AMC 荧光团。该反应经过优化，运行速度快（即 20 秒内底物转化率约 95%），因此 CypD 活性超过了肽的自发异构化。这就需要使用像 FDSS7000 (Hamamatsu Photonics) 这样的读取器，它可以在亚秒级时间尺度上同时记录来自所有板孔的信号。

Screening of the approved drugs library (single concentration = 1.25 µM) produced five hits exhibiting >50% inhibition. These included CsA and cyclosporin C, as well as topotecan, ebselen and felbamate (the last two are known to be BBB permeable). Follow-up dose response experiments confirmed their activity (Supplementary Figs 4–6) and a counter screen demonstrated that the apparent inhibition was not due to inhibition of the coupling enzyme, chymotrypsin (Fig. 4A).
对批准的药物库（单一浓度 = 1.25 µM）的筛选产生了 5 个表现出 > 50% 抑制的命中结果。其中包括 CsA 和环孢菌素 C，以及拓扑替康、依布硒啉和非氨酯（后两种已知具有 BBB 渗透性）。后续剂量反应实验证实了它们的活性（补充图 4-6），并且计数器筛选证明明显的抑制不是由于偶联酶胰凝乳蛋白酶的抑制（图 4A）。

Ebselen is an investigational drug with experimental evidence of neuroprotection, antioxidant, antiviral and anti-inflammatory effects in multiple disease models and diseases, including human stroke.^{66,68,71,72,95,96} However, the mechanisms underlying the effect of ebselen on Alzheimer’s disease-related mitochondrial dysfunction have not yet been explored. We therefore focused on ebselen in subsequent studies designed to validate the biological effects of CypD inhibition on Alzheimer’s disease- and Aβ-related mitochondrial and synaptic dysfunction and learning memory in an Alzheimer’s disease mouse model, which will greatly facilitate the development of therapeutic targets to improve mitochondrial and cognitive function for Alzheimer’s disease and/or other neurodegenerative diseases.
Ebselen 是一种在研药物，在多种疾病模型和疾病（包括人类中风）中具有神经保护、抗氧化、抗病毒和抗炎作用的实验证据。 ^{66,68,71,72,95,96} 然而，依布硒啉对阿尔茨海默病相关线粒体功能障碍影响的机制尚未被探索。因此，我们在随后的研究中重点关注依布硒啉，旨在验证 CypD 抑制对阿尔茨海默病和 Aβ 相关线粒体和突触功能障碍以及阿尔茨海默病小鼠模型学习记忆的生物效应，这将极大地促进治疗靶点的开发，以改善阿尔茨海默病的症状。阿尔茨海默病和/或其他神经退行性疾病的线粒体和认知功能。

Ebselen binding to cyclophilin D revealed by X-ray crystal structure
X 射线晶体结构揭示依布硒啉与亲环蛋白 D 的结合

We obtained the crystal structure to verify the specific binding mode of ebselen to human CypD (Supplementary Table 2). Following initial refinement, electron density that was consistent with ebselen was observed near Cys115, indicating covalent bonding to the sulphur via the selenium atom in ebselen. A similar covalent complex has been observed between ebselen and a cysteine residue in superoxide dismutase-1 (SOD1). The reaction scheme and electron density maps are shown in Fig. 4B and C. The ebselen molecule occupies a relatively neutral binding pocket environment, as depicted in Fig. 4D and does not form any hydrogen bonds with CypD residues. The overall structure of CypD-Ebselen is similar to that of apo (PDB: 4O8H) and CsA bound (PDB: 2Z6W) CypD. Root mean square deviation between Cα atoms were 0.55 Å and 0.54 Å, respectively, for 164 residues aligned using GESAMT.⁹⁷ The main differences were observed in the loop spanning residues Cys115 to Val128 and specifically those from Cys115 to Leu122 (Fig. 4E). The binding of ebselen to CypD results in movement of Cys115 by ∼1.7 Å to form the covalent S-Se bond. This perturbs the conformation of the residues between Cys115 to Leu122 relative to the apo and CsA bound structure. Although CsA binds to a similar region of CypD as ebselen, it does not alter the conformation of this loop region as it forms minimal interactions in this area. Additionally, residues Phe60 and 113 must rotate to accommodate the binding of ebselen, as shown in Fig. 4F. These results demonstrate the crystal structure of CypD in a complex with ebselen.
我们获得了晶体结构来验证依布硒啉与人 CypD 的特异性结合模式（补充表 2）。经过初步细化，在 Cys115 附近观察到与依布硒啉一致的电子密度，表明依布硒啉中的硒原子与硫发生共价键合。在依布硒啉和超氧化物歧化酶 1 (SOD1) 中的半胱氨酸残基之间观察到类似的共价复合物。反应方案和电子密度图如图4B和C所示。依布硒啉分子占据相对中性的结合袋环境，如图4D所示，并且不与CypD残基形成任何氢键。 CypD-Ebselen 的整体结构与 apo (PDB: 4O8H) 和 CsA 结合 (PDB: 2Z6W) CypD 的整体结构相似。对于使用 GESAMT 比对的 164 个残基，Cα 原子之间的均方根偏差分别为 0.55 Å 和 0.54 Å。 ⁹⁷ 主要差异是在环跨越残基Cys115至Val128，特别是从Cys115至Leu122的环跨越残基中观察到的（图4E）。 ebselen 与 CypD 的结合导致 Cys115 移动~1.7 Å，形成共价 S-Se 键。这扰乱了 Cys115 到 Leu122 之间的残基相对于 apo 和 CsA 结合结构的构象。尽管 CsA 与依布硒啉 (ebselen) 结合到 CypD 的相似区域，但它不会改变该环区域的构象，因为它在该区域形成最小的相互作用。此外，残基 Phe60 和 113 必须旋转以适应依布硒啉的结合，如图 4F 所示。这些结果证明了 CypD 与依布硒啉复合物的晶体结构。

To support the screening and crystal structure and theoretical results, we further validated the inhibitory effect of ebselen in vitro in cybrid cells and in vivo in mouse brains. The addition of ebselen to Alzheimer’s disease cybrid cells robustly diminished CypD activity by 90% (Fig. 4G). For the in vivo study, mice were given ebselen (2.5 mg/kg) through intraperitoneal injection and the CypD activity in the hippocampus was measured. The selected dose of ebselen in mice was based on the in vivo metabolism of ebselen, showing non-toxicity and biocompatibility in the brain. Detailed analysis demonstrates that up to 10% of the blood treated with ebselen enters the brain and is completely cleared within 24 h.⁹⁸ Thus, daily treatment with ebselen (2.5 mg/kg) through intraperitoneal injection, which provides a high absorption rate, maintains sufficient amounts of ebselen (IC50: ∼1.5 µM) in the brains of experimental mice. Indeed, our results indicated ∼80% reduction in CypD activity in the brain of ebselen-treated mice compared to vehicle-treated mice (Fig. 4H). This also demonstrates the presence of ebselen in the brain. Interestingly, ebselen did not alter the transcriptional and protein expression levels of CypD (Supplementary Fig. 7). These results establish that ebselen specifically and sufficiently suppresses CypD activity not only in in vitro cell-free systems but also in in vitro Alzheimer’s disease cybrid cells and in in vivo mouse brains.
为了支持筛选和晶体结构以及理论结果，我们进一步验证了依布硒啉在 cybrid 细胞的体外和小鼠大脑的体内抑制作用。在阿尔茨海默氏病 cybrid 细胞中添加依布硒啉可使 CypD 活性显着降低 90%（图 4G）。在体内研究中，通过腹腔注射给小鼠依布硒啉（2.5 mg/kg）并测量海马中的 CypD 活性。依布硒啉在小鼠体内的选择剂量是基于依布硒啉的体内代谢，在脑中显示出无毒性和生物相容性。详细分析表明，经依布硒啉处理的血液中，高达 10% 进入大脑，并在 24 小时内完全清除。 ⁹⁸ 因此，每天通过腹膜内注射依布硒啉（2.5 mg/kg）进行治疗，可提供高吸收率，从而在实验小鼠的大脑中维持足够量的依布硒啉（IC50：~1.5 µM）。事实上，我们的结果表明，与媒介物治疗的小鼠相比，依布硒啉治疗的小鼠大脑中 CypD 活性降低了约 80%（图 4H）。这也证明了依布硒啉在大脑中的存在。有趣的是，依布硒啉并没有改变 CypD 的转录和蛋白质表达水平（补充图 7）。这些结果表明，依布硒啉不仅在体外无细胞系统中特异性且充分地抑制 CypD 活性，而且在体外阿尔茨海默病 cybrid 细胞和体内小鼠大脑中也能抑制 CypD 活性。

Effect of ebselen on mPTP and mitochondrial function in Alzheimer's disease cybrid cells
依布硒啉对阿尔茨海默病cybrid细胞mPTP和线粒体功能的影响

In view of the binding of ebselen to CypD and role of CypD in mPTP function, we first assessed the effect of ebselen on mPTP opening in Alzheimer’s disease mitochondria by fluorescence-activated cell sorting (FACS) analysis of calcein signals. As shown in Fig. 5A and B, when compared with vehicle-treated Alzheimer’s disease cybrid cells, fluorescence signals were significantly increased in the presence of Co²⁺ in the ebselen-treated Alzheimer’s disease cybrid neurons, indicating that incubation with ebselen blocked mPTP opening. No significant differences between vehicle and ebselen treatment were found in the absence of Co²⁺ (Fig. 5C and D). We then examined ΔΨm using the tetramethylrhodamine (TMRM) fluorophore, whose staining intensity is an indicator of mitochondrial membrane potential. TMRM intensity was robustly decreased by 80–90% in Alzheimer’s disease cybrids compared to non-Alzheimer’s disease cybrid cells, whereas decreased TMRM signals were largely rescued by ebselen treatment (Fig. 5E and F). Furthermore, ebselen nearly abolished Aβ-mediated ROS overproduction as measured by a highly specific and sensitive electron paramagnetic resonance (EPR) (Fig. 5G and H). These results indicate that inhibition of CypD by ebselen prevents mPTP opening and improves mitochondrial membrane potential in Alzheimer’s disease cybrid cells.
鉴于依布硒啉与CypD的结合以及CypD在mPTP功能中的作用，我们首先通过钙黄绿素信号的荧光激活细胞分选（FACS）分析评估了依布硒啉对阿尔茨海默病线粒体中mPTP开放的影响。如图 5A 和 B 所示，与媒介物处理的阿尔茨海默病 cybrid 细胞相比，在依布硒啉处理的阿尔茨海默病 cybrid 神经元中，在 Co ²⁺ 存在的情况下，荧光信号显着增加，表明孵育用依布硒啉阻断 mPTP 开放。在没有 Co ²⁺ 的情况下，媒介物和依布硒啉治疗之间没有发现显着差异（图 5C 和 D）。然后，我们使用四甲基罗丹明 (TMRM) 荧光团检查 ΔΨm，其染色强度是线粒体膜电位的指标。与非阿尔茨海默病 cybrid 细胞相比，阿尔茨海默病 cybrid 细胞的 TMRM 强度显着降低 80-90%，而依布硒啉治疗很大程度上可以挽救降低的 TMRM 信号（图 5E 和 F）。此外，通过高度特异性和灵敏的电子顺磁共振（EPR）测量，依布硒啉几乎消除了Aβ介导的ROS过量产生（图5G和H）。这些结果表明，依布硒啉对 CypD 的抑制可防止 mPTP 打开并改善阿尔茨海默病 cybrid 细胞中的线粒体膜电位。

In view of the strong association of mPTP formation with mitochondrial dysfunction, we next assessed whether inhibition mPTP by ebselen could restore Alzheimer’s disease-affected mitochondrial respiration and glycolysis in Alzheimer’s disease cybrid cells. Indeed, we observed that ebselen treatment significantly increased basal and maximal OCR, ATP-linked OCR, and mitochondrial ATP production compared to vehicle-treated Alzheimer’s disease cybrid cells (Fig. 5I–M). Furthermore, ebselen improved mitochondrial respiration in a dose-dependent manner, which rules out potential off-target activity (Fig. 5N–Q). Similarly, glycolytic capacity, including ECAR, basal, PER and glycolytic ATP levels were significantly elevated in ebselen-treated Alzheimer’s disease cybrid cells (Fig. 6A–F). These protective effects were dose-dependent at micromolar concentrations of ebselen (range 1–2.5 µM, Fig. 6G–K). These data demonstrate the protective effect of ebselen against Alzheimer’s disease-related mitochondrial and glycolytic dysfunction and bioenergetics.
鉴于 mPTP 形成与线粒体功能障碍密切相关，我们接下来评估了依布硒啉抑制 mPTP 是否可以恢复阿尔茨海默病 cybrid 细胞中受阿尔茨海默病影响的线粒体呼吸和糖酵解。事实上，我们观察到，与媒介物处理的阿尔茨海默病细胞杂种细胞相比，依布硒啉处理显着增加了基础和最大 OCR、ATP 相关 OCR 和线粒体 ATP 产量（图 5I-M）。此外，依布硒啉以剂量依赖性方式改善线粒体呼吸，这排除了潜在的脱靶活性（图5N-Q）。同样，在依布硒啉处理的阿尔茨海默氏病细胞中，糖酵解能力，包括 ECAR、基础、PER 和糖酵解 ATP 水平显着升高（图 6A-F）。这些保护作用在依布硒啉微摩尔浓度下呈剂量依赖性（范围1-2.5 µM，图6G-K）。这些数据证明了依布硒啉对阿尔茨海默病相关的线粒体和糖酵解功能障碍以及生物能的保护作用。

Effect of ebselen on cyclophilin D-mediated mitochondrial dysfunction
依布硒啉对亲环蛋白 D 介导的线粒体功能障碍的影响

To verify the specific inhibitory effect of ebselen on CypD-mediated mitochondrial defect, cybrid cells were transfected with the pcDNA3-hCypD construct to overexpress human CypD in the presence or absence of ebselen. Immunoblotting confirmed increased expression of CypD in pcDNA3-hCypD transfected cells compared with control cells transfected with pcDNA3 vector (Fig. 7A). CypD-overexpressing cybrid cells exhibited significantly reduced OCR and ATP levels as compared with pcDNA3 vector transfected cybrids, while these detrimental effects were completed blocked by the addition of ebselen (Fig. 7B–E). Thus, the protective effect of ebselen on aberrant Alzheimer’s disease mitochondrial function is achieved by targeting CypD.
为了验证依布硒啉对 CypD 介导的线粒体缺陷的特异性抑制作用，用 pcDNA3-hCypD 构建体转染 cybrid 细胞，以在存在或不存在依布硒啉的情况下过表达人 CypD。免疫印迹证实与用pcDNA3载体转染的对照细胞相比，pcDNA3-hCypD转染的细胞中CypD的表达增加(图7A)。与 pcDNA3 载体转染的细胞杂种相比，CypD 过表达的细胞杂种细胞表现出显着降低的 OCR 和 ATP 水平，而这些有害影响可通过添加依布硒啉完全阻断（图 7B-E）。因此，依布硒啉对异常阿尔茨海默病线粒体功能的保护作用是通过靶向 CypD 来实现的。

Ebselen improves mitochondrial and synaptic function in an Aβ/Alzheimer's disease mice
Ebselen 改善 Aβ/阿尔茨海默病小鼠的线粒体和突触功能

To assess the effect of ebselen on mitochondrial function in an Alzheimer’s disease mouse model, mice at 11 months of age were administered ebselen (2.5 mg/kg, daily, i.p. injection) for 8 weeks. Mitochondrial respiration was evaluated by assessing complex IV activity and ATP levels. Complex IV activity and ATP levels were significantly increased in ebselen-treated mAPP mice compared to vehicle-treated mice (Fig. 8A and B). Ebselen treatment also blunted H₂O₂ production in mAPP mice (Fig. 8C). These results demonstrate that administration of ebselen attenuates Aβ-induced mitochondrial dysfunction and disruption of energy metabolism and suppresses oxidative stress in Alzheimer’s disease mice with amyloid pathology.
为了评估依布硒啉对阿尔茨海默病小鼠模型线粒体功能的影响，对 11 个月大的小鼠施用依布硒啉（2.5 毫克/公斤，每天，腹腔注射）8 周。通过评估复合物 IV 活性和 ATP 水平来评估线粒体呼吸。与媒介物处理的小鼠相比，依布硒啉处理的 mAPP 小鼠的复合物 IV 活性和 ATP 水平显着增加（图 8A 和 B）。 Ebselen 治疗还削弱了 mAPP 小鼠中 H ₂ O ₂ 的产生（图 8C）。这些结果表明，依布硒啉可减轻 Aβ 诱导的线粒体功能障碍和能量代谢的破坏，并抑制患有淀粉样蛋白病理的阿尔茨海默病小鼠的氧化应激。

Given the importance of synaptic mitochondrial function for synaptic transmission, we evaluated the potential role of ebselen in Aβ-induced synaptic dysfunction by recording LTP in hippocampal CA1 neurons from wild-type mice exposed to oligomeric Aβ. Hippocampal neurons in slices exposed to oligomeric Aβ (200 nM) displayed a significant decrease in LTP from baseline to 135%, whereas LTP was almost completely recovered in the presence of ebselen (Fig. 8D–F). Basal synaptic transmission (BST) was not significantly changed, as shown by field-excitatory post-synaptic potential (fEPSPs) and LTP between vehicle- and ebselen-treated hippocampal slices (Fig. 8G). These results indicate that ebselen attenuates Aβ-induced synaptic dysfunction.
鉴于突触线粒体功能对突触传递的重要性，我们通过记录暴露于寡聚 Aβ 的野生型小鼠海马 CA1 神经元的 LTP 来评估依布硒啉在 Aβ 诱导的突触功能障碍中的潜在作用。暴露于寡聚 Aβ (200 nM) 的切片中的海马神经元的 LTP 从基线显着下降至 135%，而在依布硒啉存在下，LTP 几乎完全恢复（图 8D-F）。基础突触传递（BST）没有显着改变，如媒介物和依布硒啉处理的海马切片之间的场兴奋性突触后电位（fEPSPs）和LTP所示（图8G）。这些结果表明依布硒啉可减轻 Aβ 诱导的突触功能障碍。

Inhibition of cyclophilin D by ebselen improves learning and memory in Alzheimer’s disease mice
依布硒啉抑制亲环蛋白 D 可改善阿尔茨海默病小鼠的学习和记忆

We next assessed whether the protective effects of ebselen against aberrant synaptic dysfunction were reflected in changes in learning and memory behaviour in 5xFAD mice, another Alzheimer’s disease mouse model expressing/accumulating human Aβ in the cortex and mitochondria. 5xFAD mice were treated with ebselen at 6 months of age. After 8 weeks of treatment, mice were subjected to the Morris water maze (MWM) test, one of the most widely used tasks to assess their spatial learning and memory in relation to hippocampal function. Alzheimer’s disease mice exhibited a significantly longer latency to locate the hidden platform during the training session (Fig. 8H), decreased the number of times crossing the target (Fig. 8I) and decreased time spent in the target quadrant (Fig. 8J and K) during the training period, compared to nonTg mice. Notably, ebselen treatment almost fully restored learning and memory, as shown by significantly shorter latency to find the platform during training and an increase in the number of target crossings and the time spent in the target quadrant during the probe trial (Fig. 8I–K). The swimming speeds were comparable among the four groups of mice (Fig. 8L), indicating that the improvement in learning and memory was not due to alteration in motor activity. These results indicate that the blockade of CypD by ebselen improves synaptic and cognitive function.
接下来，我们评估了依布硒啉对异常突触功能障碍的保护作用是否反映在 5xFAD 小鼠的学习和记忆行为变化中，5xFAD 小鼠是另一种在皮质和线粒体中表达/积累人类 Aβ 的阿尔茨海默病小鼠模型。 5xFAD 小鼠在 6 个月大时接受依布硒啉治疗。治疗 8 周后，小鼠接受莫里斯水迷宫 (MWM) 测试，这是评估其与海马功能相关的空间学习和记忆的最广泛使用的任务之一。阿尔茨海默病小鼠在训练过程中表现出明显更长的潜伏期来定位隐藏平台（图8H），减少穿过目标的次数（图8I）并减少在目标象限中花费的时间（图8J和K） ）在训练期间，与非Tg小鼠相比。值得注意的是，依布硒啉治疗几乎完全恢复了学习和记忆，如训练期间寻找平台的潜伏期显着缩短以及探索试验期间目标交叉次数和在目标象限中花费的时间增加所示（图8I-K） ）。四组小鼠的游泳速度相当（图8L），这表明学习和记忆的改善并不是由于运动活动的改变。这些结果表明依布硒啉对 CypD 的阻断可改善突触和认知功能。

Ebselen suppresses non-inflammatory mediators in the brain of Alzheimer’s disease mice
Ebselen 抑制阿尔茨海默病小鼠大脑中的非炎症介质

Because mitochondria play an important role in proinflammatory signalling, damaged mitochondria could serve as endogenous danger signals that potentiate inflammation through production of ROS and/or damage-associated molecular patterns (DAMPs), we thereby propose that blockade of Aβ-induced mitochondrial dysfunction by CypD inhibitor ebselen may lessen inflammation in Aβ-affected brain. To test this concept, we assessed expression levels of proinflammatory cytokines and chemokines in the cortices of Alzheimer’s disease mice. Consistent with our previous studies,^99,100 Aβ/Alzheimer’s disease mice showed a significant increase in cytokines (TNF-α and IL-1β) and chemokine MCP-1 compared with nonTg control mice. Notably, ebselen-treated mice exhibited largely attenuated the production of these proinflammatory mediators (Supplementary Fig. 8). These results suggest a possible link between CypD-mediated mitochondria dysfunction and neuroinflammation contributing to synaptic injury and cognitive decline relevant to Aβ/Alzheimer’s disease.
由于线粒体在促炎信号传导中发挥重要作用，受损的线粒体可以作为内源性危险信号，通过产生 ROS 和/或损伤相关分子模式 (DAMP) 来加剧炎症，因此我们建议 CypD 阻断 Aβ 诱导的线粒体功能障碍抑制剂 ebselen 可能会减轻 Aβ 影响的大脑中的炎症。为了检验这个概念，我们评估了阿尔茨海默病小鼠皮质中促炎细胞因子和趋化因子的表达水平。与我们之前的研究一致，与非 Tg 对照小鼠相比， ^99,100 Aβ/阿尔茨海默病小鼠的细胞因子（TNF-α 和 IL-1β）和趋化因子 MCP-1 显着增加。值得注意的是，依布硒啉治疗的小鼠表现出在很大程度上减弱了这些促炎介质的产生（补充图8）。这些结果表明 CypD 介导的线粒体功能障碍与神经炎症之间可能存在联系，神经炎症会导致与 Aβ/阿尔茨海默病相关的突触损伤和认知能力下降。

Discussion 讨论

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease and could drive or mediate various Alzheimer’s disease-related pathologies. In the present study, we comprehensively analysed how modulation of CypD affects mitochondrial respiration and glycolytic bioenergetics in sporadic Alzheimer’s disease mitochondria and the effect of mitochondrial CypD blockade on mitochondrial and glycolytic function and energy metabolism. These experiments were performed using human neuronal cybrids with Alzheimer’s disease-derived mitochondria as an ex vivo model of mitochondrial defects in human sporadic Alzheimer’s disease and two Alzheimer’s disease mouse models. Furthermore, we identified a novel CypD inhibitor, ebselen, whose interaction with CypD has been confirmed by crystallography.
线粒体功能障碍是阿尔茨海默病的早期病理特征，可能驱动或介导各种与阿尔茨海默病相关的病理。在本研究中，我们全面分析了CypD的调节如何影响散发性阿尔茨海默病线粒体中的线粒体呼吸和糖酵解生物能，以及线粒体CypD阻断对线粒体和糖酵解功能以及能量代谢的影响。这些实验是使用具有阿尔茨海默病衍生线粒体的人类神经元细胞杂种作为人类散发性阿尔茨海默病和两种阿尔茨海默病小鼠模型的线粒体缺陷的离体模型进行的。此外，我们还发现了一种新型 CypD 抑制剂 ebselen，其与 CypD 的相互作用已通过晶体学得到证实。

First, our results demonstrate a significant upregulation of CypD in Alzheimer’s disease cybrid cells compared to nonAD controls, in agreement with observations of elevated CypD levels in human Alzheimer’s disease patients and in transgenic Alzheimer’s disease mouse models with amyloid pathology.⁴⁴ Increased expression of CypD occurs in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease^44,101,102 and diabetes^103,104 and leads to mitochondrial perturbation. CypD expression levels are further elevated in Aβ-enriched diabetic brains compared to non-diabetic Aβ/Alzheimer’s disease mice, and are associated with memory impairment. Higher ROS levels lead to deleterious effects, including opening of the mPTP and consequent blunting of mitochondrial function and upregulation of CypD expression.¹⁰⁵ Genetic or pharmacological inhibition of CypD attenuates ROS-induced mitochondrial dysfunction,^105,106 suggesting a link between ROS and the modulation of CypD activity under pathological conditions, such as accumulation of toxic metabolites in Aβ/tau-rich Alzheimer’s disease brains¹⁰⁷ and Alzheimer’s disease cybrids with higher levels of ROS and impaired mPTP/mitochondrial function. Thus, Alzheimer’s disease-related mitochondrial dysfunction or Aβ could directly or indirectly stimulate CypD activity through increased ROS levels. In fact, loss-of-function mutant CypD without PPIase activity is not able to regulate mPTP and related mitochondrial function,¹⁰⁸ suggesting that CypD activity facilitates mPTP activation. Indeed, blockade of CypD with its inhibitors (ebselen or CsA) or post-transcriptional gene silencing by targeting Ppif reversed CypD-mediated mPTP opening and mitochondrial and glycolytic perturbations. Accordingly, scavenging mitochondrial ROS by application of a mitochondria targeted antioxidant (MitoTEMPO) alleviated mitochondrial and glycolytic perturbation. Additionally, post-translational modifications, such as acetylation and phosphorylation, are known to exert regulatory effects on CypD activity and consequently on mPTP opening.^108-111
首先，我们的结果表明，与非 AD 对照相比，阿尔茨海默病 cybrid 细胞中 CypD 显着上调，这与人类阿尔茨海默病患者和具有淀粉样蛋白病理的转基因阿尔茨海默病小鼠模型中 CypD 水平升高的观察结果一致。 ⁴⁴ CypD 表达增加发生在神经退行性疾病中，包括阿尔茨海默病、帕金森病、亨廷顿病 ^44,101,102 和糖尿病 ^103,104 ，并导致线粒体扰动。与非糖尿病 Aβ/阿尔茨海默病小鼠相比，富含 Aβ 的糖尿病小鼠大脑中 CypD 表达水平进一步升高，并且与记忆障碍相关。较高的 ROS 水平会导致有害影响，包括打开 mPTP 以及随之而来的线粒体功能减弱和 CypD 表达上调。 ¹⁰⁵ CypD 的遗传或药理学抑制可减轻 ROS 诱导的线粒体功能障碍， ^105,106 表明 ROS 与病理条件下 CypD 活性的调节之间存在联系，例如 Aβ 中有毒代谢物的积累富含 /tau 的阿尔茨海默病大脑 ¹⁰⁷ 和具有较高 ROS 水平和受损 mPTP/线粒体功能的阿尔茨海默病杂种。因此，阿尔茨海默病相关的线粒体功能障碍或 Aβ 可以通过增加 ROS 水平直接或间接刺激 CypD 活性。事实上，没有 PPIase 活性的功能丧失突变体 CypD 无法调节 mPTP 和相关的线粒体功能， ¹⁰⁸ 表明 CypD 活性促进 mPTP 激活。 事实上，用其抑制剂（依布硒啉或 CsA）阻断 CypD 或通过靶向 Ppif 进行转录后基因沉默可逆转 CypD 介导的 mPTP 打开以及线粒体和糖酵解扰动。因此，通过应用线粒体靶向抗氧化剂（MitoTEMPO）清除线粒体ROS可以减轻线粒体和糖酵解的扰动。此外，已知翻译后修饰（例如乙酰化和磷酸化）对 CypD 活性产生调节作用，从而对 mPTP 打开产生调节作用。 ^108-111

Second, we demonstrate a link between CypD-mediated mitochondrial respiration and glycolytic energy metabolism associated with mitochondrial defects in Alzheimer’s disease. Cytosolic glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle are responsible for the metabolism of a majority of the glucose entering brain cells and the generation of ATP needed to support brain function. Neurons have a high demand for ATP for synaptic activity, neurotransmission and memory consolidation. Although glycolysis produces lower ATP yields than mitochondrial oxidative phosphorylation, it provides more quickly accessible amounts of ATP, particularly at the plasma membrane and synaptic vesicles.¹¹² The advantage of glycolysis over mitochondrial oxidative phosphorylation is at least 2-fold faster generation of ATP.¹¹³ Lower rates of glycolysis and higher brain glucose levels are associated with more severe amyloid and tau pathology in the brains of Alzheimer’s disease patients. More severe reductions in brain glycolysis were also associated with expression of Alzheimer’s disease symptoms.⁹¹ Neurons have the ability to increase their own glycolysis in response to stimuli for rapid energy replenishment. However, in our study, neuronal Alzheimer’s disease cybrids exhibited reduced glycolytic energy and mitochondrial respiration. Intriguingly, both CypD inhibitors (CsA or ebselen) and knockdown of CypD enhanced glycolytic bioenergetics, suggesting that CypD plays an important role in glycolysis as well as mitochondrial respiratory function. In view of the protection of scavenging mitochondrial ROS on Alzheimer’s disease glycolytic defects (Fig. 3), ROS could represent a mechanistic link to Alzheimer’s disease-related glycolytic impairment. Other oxidative stress-related signalling pathways, such as mitogen-activated protein kinase and related activation of transcription factor NFKB,^{51,61,80,114-117} may also be involved in glycolytic metabolism. In the near future, exploration of the Alzheimer’s disease-related metabolic interplay between mitochondrial oxidation and glycolytic exhaustion will be required.
其次，我们证明了 CypD 介导的线粒体呼吸和与阿尔茨海默病线粒体缺陷相关的糖酵解能量代谢之间的联系。胞质糖酵解和线粒体三羧酸 (TCA) 循环负责进入脑细胞的大部分葡萄糖的代谢以及支持脑功能所需的 ATP 的生成。神经元对 ATP 的突触活动、神经传递和记忆巩固有很高的需求。尽管糖酵解产生的 ATP 产量低于线粒体氧化磷酸化，但它提供了更快速可利用的 ATP 量，特别是在质膜和突触小泡处。 ¹¹² 糖酵解相对于线粒体氧化磷酸化的优势是 ATP 生成速度至少快 2 倍。 ¹¹³ 较低的糖酵解率和较高的脑葡萄糖水平与阿尔茨海默病患者大脑中更严重的淀粉样蛋白和 tau 蛋白病理有关。大脑糖酵解更严重的减少也与阿尔茨海默病症状的表达有关。 ⁹¹ 神经元能够响应刺激而增加自身的糖酵解，从而快速补充能量。然而，在我们的研究中，神经元阿尔茨海默病细胞杂种表现出糖酵解能量和线粒体呼吸减少。有趣的是，CypD 抑制剂（CsA 或依布硒啉）和 CypD 敲低均增强了糖酵解生物能，表明 CypD 在糖酵解以及线粒体呼吸功能中发挥着重要作用。鉴于清除线粒体 ROS 对阿尔茨海默病糖酵解缺陷的保护作用（图 3），ROS 可能代表与阿尔茨海默病相关的糖酵解障碍的机制联系。 其他氧化应激相关信号通路，如丝裂原激活蛋白激酶和转录因子NFKB的相关激活， ^{51,61,80,114-117} 也可能参与糖酵解代谢。在不久的将来，将需要探索线粒体氧化和糖酵解耗竭之间与阿尔茨海默病相关的代谢相互作用。

Third, we identified a new class of CypD inhibitors by screening known pharmacologically active compounds. Ebselen is a covalent inhibitor that inactivates many enzymes by modifying functionally relevant cysteine residues. While this polypharmacology may pose challenges for drug development, it does not preclude a therapeutic effect. We further confirmed the binding mode of the selected inhibitor ebselen to CypD using X-ray crystallography. Ebselen interacts with Cys115 in CypD to form a covalent S-Se bond, perturbing the conformation of the residues between Cys115 and Leu122 relative to the apo and CsA-bound structure. Although CsA binds to a similar region of CypD, it does not alter the conformation of this loop region. This observation, paired with the larger macrocyclic structure of CsA, suggests that the precise mechanism of inhibition differs between the two inhibitors. A series of macrocyclic CypD inhibitors was recently described that bind to the active site and but also span the neighbouring S2 pocket.¹¹⁸ Ebselen, which is a much smaller scaffold relative to a macrocyclic compound, binds in the active site region of CypD via covalent attachment with Cys115. As such, one can envision that ebselen could serve as the basis for chemical expansion to develop more potent inhibitors.
第三，我们通过筛选已知的药理活性化合物确定了一类新的 CypD 抑制剂。 Ebselen 是一种共价抑制剂，通过修饰功能相关的半胱氨酸残基来灭活许多酶。虽然这种多药理学可能给药物开发带来挑战，但它并不排除治疗效果。我们使用X射线晶体学进一步证实了所选抑制剂依布硒啉与CypD的结合模式。 Ebselen 与 CypD 中的 Cys115 相互作用形成共价 S-Se 键，扰乱 Cys115 和 Leu122 之间的残基相对于 apo 和 CsA 结合结构的构象。尽管 CsA 与 CypD 的相似区域结合，但它不会改变该环区域的构象。这一观察结果与 CsA 较大的大环结构相结合，表明两种抑制剂的精确抑制机制不同。最近描述了一系列大环 CypD 抑制剂，它们与活性位点结合，但也跨越邻近的 S2 口袋。 ¹¹⁸ Ebselen 是一种比大环化合物小得多的支架，通过与 Cys115 共价连接结合在 CypD 的活性位点区域。因此，人们可以设想依布硒啉可以作为化学扩张的基础来开发更有效的抑制剂。

Functionally, ebselen can specifically inhibit CypD activity in Alzheimer’s disease-derived mitochondria in vitro and mouse brains in vivo and in an enzyme kinetic assay using human CypD protein in an in vitro cell-free system. Importantly, ebselen is able to block Alzheimer’s disease-related mPTP opening and attenuates mitochondrial dysfunction by elevating oxygen consumption rate, glycolytic capacity and ATP levels in Alzheimer’s disease-derived mitochondria. Furthermore, ebselen attenuated Aβ-induced mitochondrial and synaptic dysfunction and improved learning and memory in Alzheimer’s disease mice. Ebselen reverses CypD-mediated mitochondrial dysfunction in cybrid cells expressing human CypD, supporting that the effect of ebselen is dependent on CypD inhibition. We noted that rescue of mitochondrial OCR in cultured Alzheimer’s disease cybrid cells occurred at ebselen concentration lower than the IC50 of CypD measured in cell-free systems (∼0.5 µM versus ∼1.2 µM), suggesting that alternative mechanisms or co-factors could contribute to ebselen-mediated protective effects on mitochondrial respiration. Differences in IC50 between in vitro cell-free system and in vitro cell cultures, along with variations in substrate concentrations, may also account for the observed results. Nevertheless, our results showing binding of ebselen to CypD by crystallography, specific inhibition of CypD activity and reversal of CypD-mediated mitochondrial dysfunction, support CypD as one of the targets of ebselen. Interestingly, ebselen also significantly reduced the inflammatory response in Alzheimer’s disease mice brains. Thus, it is quite possible that ebselen improves mitochondrial and cognitive function through several pathways by blocking mPTP-induced mitochondrial dysfunction, subsequently reducing mitochondrial oxidative stress, restoring synaptic activity and suppressing neuroinflammation. To support this, ebselen has been reported as a neuroprotective, anti-neuroinflammatory and antioxidant compound in several neurological diseases, including stroke, hearing loss, Meniere’s disease and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patient^119-122 and in other Alzheimer’s disease mouse models.^75,77,123 Compared to existing CypD inhibitors with severe side effects and poor BBB permeability, ebselen had no apparent toxicity and is able to cross the BBB.⁷⁰ ebselen was well tolerated, safe and effective in phase I–III clinical trials.^64,66,67 Indeed, we found no significant toxicity, including mitochondrial and synaptic activity, in both cellular and mouse models. Hence, these data suggest that ebselen is a potential candidate for a clinically useful CypD inhibitor to rescue and/or enhance mitochondrial and cognitive function.
从功能上讲，依布硒啉可以在体外特异性抑制阿尔茨海默病来源的线粒体和体内小鼠大脑中的 CypD 活性，并在体外无细胞系统中使用人 CypD 蛋白进行酶动力学测定。重要的是，依布硒啉能够阻断阿尔茨海默病相关的 mPTP 打开，并通过提高阿尔茨海默病来源的线粒体中的耗氧率、糖酵解能力和 ATP 水平来减轻线粒体功能障碍。此外，依布硒啉还可减轻 Aβ 诱导的线粒体和突触功能障碍，并改善阿尔茨海默病小鼠的学习和记忆能力。 Ebselen 可逆转表达人 CypD 的 cybrid 细胞中 CypD 介导的线粒体功能障碍，这支持依布硒啉的作用依赖于 CypD 抑制。我们注意到，在培养的阿尔茨海默病 cybrid 细胞中，依布硒啉浓度低于在无细胞系统中测量的 CypD 的 IC50（~0.5 µM 与~1.2 µM）时，线粒体 OCR 得到拯救，这表明替代机制或辅助因子可能有助于依布硒啉介导的对线粒体呼吸的保护作用。体外无细胞系统和体外细胞培养物之间 IC50 的差异以及底物浓度的变化也可能解释了观察到的结果。尽管如此，我们的结果显示，通过晶体学，依布硒啉与 CypD 结合，对 CypD 活性的特异性抑制以及 CypD 介导的线粒体功能障碍的逆转，支持 CypD 作为依布硒啉的靶标之一。有趣的是，依布硒啉还显着降低了阿尔茨海默病小鼠大脑的炎症反应。 因此，依布硒啉很可能通过阻断 mPTP 诱导的线粒体功能障碍、随后减少线粒体氧化应激、恢复突触活性和抑制神经炎症，通过多种途径改善线粒体和认知功能。为了支持这一点，依布硒啉已被报道为一种神经保护、抗神经炎症和抗氧化化合物，可治疗多种神经系统疾病，包括中风、听力损失、梅尼埃病和严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 患者 ^119-122 以及其他阿尔茨海默病小鼠模型。 ^75,77,123 与现有副作用严重、血脑屏障通透性差的CypD抑制剂相比，依布硒啉无明显毒性，且能够穿过血脑屏障。 ⁷⁰ 依布硒啉在I-III期临床试验中耐受​​性良好、安全有效。 ^64,66,67 事实上，我们在细胞和小鼠模型中没有发现明显的毒性，包括线粒体和突触活性。因此，这些数据表明依布硒啉是临床上有用的 CypD 抑制剂的潜在候选者，可挽救和/或增强线粒体和认知功能。

Mitochondrial and synaptic dysfunction and inflammation co-exist in Alzheimer’s disease patients as key contributors to Alzheimer’s disease pathophysiology. It is essential and logical to develop multi-targeted drugs to effectively treat and/or halt the progression of neurodegeneration and Alzheimer’s disease. Mitochondria function as signalling platforms in innate immune and proinflammatory responses. Dysfunctional mitochondria could be the source of internally produced DAMPs, which functions as danger signals, leading to the sterile inflammation seen in the Alzheimer’s disease brain. Mitochondria, thereby, could drive many Alzheimer’s disease pathologies and are the mechanistic link between mitochondrial and synaptic pathology and neuroinflammation. We propose that mitochondria hold potential as multi-action therapeutic target for Alzheimer’s disease at both an early stage before amyloid pathology and/or even at later stages with significant amyloid/tau pathology to slow progression of the disease. Targeting mitochondria with CypD inhibitor to reverse neurodegeneration and cognitive decline represents a unique multifunctional agent with important protective effects on mitochondrial bioenergetics linked to synaptic transmission and inflammation. Our results indicate that inhibition of CypD exerts multi-protective actions by boosting mitochondrial biogenetics, suppressing oxidative stress and inflammatory response, and improving synaptic and cognitive function relevant to Alzheimer’s disease pathogenesis. Moreover, there is evidence that ebselen inhibits the activity of acetylcholinesterase (AChE).¹²⁴ AChE inhibitors, such as donepezil and galantamine, have been used to treat cognitive deficits, mainly in Alzheimer’s disease patients. This observation strengthens the translational significance of Ebselen’s benefits as a potential multi-target therapeutic for Alzheimer’s disease.
线粒体和突触功能障碍以及炎症在阿尔茨海默病患者中共存，是阿尔茨海默病病理生理学的关键因素。开发多靶点药物来有效治疗和/或阻止神经退行性疾病和阿尔茨海默病的进展是必要且合乎逻辑的。线粒体在先天免疫和促炎症反应中充当信号平台。功能失调的线粒体可能是内部产生 DAMP 的来源，DAMP 充当危险信号，导致阿尔茨海默病患者大脑出现无菌性炎症。因此，线粒体可以驱动许多阿尔茨海默氏病病理，并且是线粒体和突触病理与神经炎症之间的机制联系。我们认为，线粒体有潜力作为阿尔茨海默氏病的多作用治疗靶点，无论是在淀粉样蛋白病理发生之前的早期阶段，还是在具有显着淀粉样蛋白/tau蛋白病理发生的晚期阶段，以减缓疾病的进展。用 CypD 抑制剂靶向线粒体来逆转神经退行性变和认知能力下降是一种独特的多功能药物，对与突触传递和炎症相关的线粒体生物能具有重要的保护作用。我们的研究结果表明，抑制 CypD 可通过促进线粒体生物遗传学、抑制氧化应激和炎症反应以及改善与阿尔茨海默病发病机制相关的突触和认知功能来发挥多重保护作用。此外，有证据表明依布硒啉可抑制乙酰胆碱酯酶（AChE）的活性。 ¹²⁴ 乙酰胆碱酯酶抑制剂，例如多奈哌齐和加兰他敏，已被用于治疗认知缺陷，主要用于阿尔茨海默病患者。 这一观察结果强化了 Ebselen 作为阿尔茨海默病潜在多靶点治疗药物的转化意义。

In summary, the present study establishes a new link between ebselen and CypD involved in mitochondrial function and energy metabolism associated with sporadic Alzheimer’s disease- and Aβ-mediated aberrant mitochondria. Blockade of CypD may be a promising target focusing on the role of the mitochondrial network in Alzheimer’s disease, which offers a new avenue for therapeutic discovery in Alzheimer’s disease, such as mitochondrial enhancers and/or CypD/mPTP blockers. Ebselen derivatives or mimics with high potency and less toxicity also hold potential for the treatment of neurodegenerative diseases, such as Alzheimer’s disease, in the near future. Hence, these findings will motivate researchers in the fields of mitochondrial medicine and Alzheimer’s disease to develop multi-targeted drugs to effectively reverse neurodegeneration and synaptic injury, control neuroinflammation and improve cognition.
总之，本研究在 ebselen 和 CypD 之间建立了新的联系，参与与散发性阿尔茨海默病和 Aβ 介导的异常线粒体相关的线粒体功能和能量代谢。 CypD 的阻断可能是一个有前途的靶点，重点关注线粒体网络在阿尔茨海默病中的作用，这为阿尔茨海默病的治疗发现提供了新途径，例如线粒体增强剂和/或 CypD/mPTP 阻断剂。依布硒啉衍生物或类似物具有高效、低毒的特点，在不久的将来也有望用于治疗神经退行性疾病，例如阿尔茨海默病。因此，这些发现将激励线粒体医学和阿尔茨海默病领域的研究人员开发多靶点药物，以有效逆转神经退行性变和突触损伤，控制神经炎症并改善认知。

Data availability 数据可用性

Data supporting the findings of this study are available upon reasonable request from the corresponding author.
支持本研究结果的数据可根据相应作者的合理要求提供。

Acknowledgements 致谢

We thank David Wilson for assisting with CypD assay development in HTS assay and screening, Valasani Koteswara Rao for the preparation of crystal complex of ebselen with CypD protein for crystal analysis, and Michael Kissner for assisting with flow cytometry. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Thank Gladstone for providing mAPP mice (J-20 line) and MMRC of Jackson Laboratory for 5xFAD mice for this study.
我们感谢 David Wilson 协助 HTS 测定和筛选中的 CypD 测定开发，感谢 Valasani Koteswara Rao 制备依布硒啉与 CypD 蛋白的晶体复合物以进行晶体分析，感谢 Michael Kissner 协助流式细胞术。通过与 Hauptman-Woodward 医学研究所签订的合同，工业高分子晶体学协会的公司支持在先进光子源使用 IMCA-CAT 光束线 17-ID。先进光子源的使用得到了美国能源部、科学办公室、基础能源科学办公室的支持，合同号为 DE-AC02-06CH11357。感谢 Gladstone 为本研究提供 mAPP 小鼠（J-20 系）和 Jackson 实验室的 MMRC 5xFAD 小鼠。

Funding 资金

This work was supported by grants from the National Institute on Aging (R37AG037319, R01AG069426, 1RF1AG077848 and P30AG035982).
这项工作得到了国家老龄化研究所 (R37AG037319、R01AG069426、1RF1AG077848 和 P30AG035982) 的资助。

Competing interests 利益争夺

The authors report no competing interests.
作者报告没有竞争利益。

Supplementary material 补充材料

Supplementary material is available at Brain online.
补充材料可在 Brain 在线获取。

References 参考