mTOR 在正常衰老过程中驱动脑血管、突触和认知功能障碍

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mTOR drives cerebrovascular, synaptic, and cognitive dysfunction in normative aging

Abstract

摘要

Cerebrovascular dysfunction and cognitive decline are highly prevalent in aging, but the mechanisms underlying these impairments are unclear. Cerebral blood flow decreases with aging and is one of the earliest events in the pathogenesis of Alzheimer’s disease (AD). We have previously shown that the mechanistic/mammalian target of rapamycin (mTOR) drives disease progression in mouse models of AD and in models of cognitive impairment associated with atherosclerosis, closely recapitulating vascular cognitive impairment. In the present studies, we sought to determine whether mTOR plays a role in cerebrovascular dysfunction and cognitive decline during normative aging in rats. Using behavioral tools and MRI‐based functional imaging, together with biochemical and immunohistochemical approaches, we demonstrate that chronic mTOR attenuation with rapamycin ameliorates deficits in learning and memory, prevents neurovascular uncoupling, and restores cerebral perfusion in aged rats. Additionally, morphometric and biochemical analyses of hippocampus and cortex revealed that mTOR drives age‐related declines in synaptic and vascular density during aging. These data indicate that in addition to mediating AD‐like cognitive and cerebrovascular deficits in models of AD and atherosclerosis, mTOR drives cerebrovascular, neuronal, and cognitive deficits associated with normative aging. Thus, inhibitors of mTOR may have potential to treat age‐related cerebrovascular dysfunction and cognitive decline. Since treatment of age‐related cerebrovascular dysfunction in older adults is expected to prevent further deterioration of cerebral perfusion, recently identified as a biomarker for the very early (preclinical) stages of AD, mTOR attenuation may potentially block the initiation and progression of AD.

脑血管功能障碍和认知能力下降在老年人中非常普遍,但是这些损伤的潜在机制尚不清楚。脑血流量随着年龄的增长而减少,是阿尔茨海默病(AD)发病机制中最早的事件之一。我们先前已经证明,雷帕霉素的机械/哺乳动物靶蛋白(mTOR)在 AD 小鼠模型和动脉粥样硬化相关的认知损害模型中,促进疾病的进展,这与血管性认知损害密切相关。在目前的研究中,我们试图确定 mTOR 是否在大鼠正常老化过程中的脑血管功能障碍和认知能力下降中起作用。采用行为学方法和 MRI 功能成像技术,结合生物化学和免疫组织化学方法,证明雷帕霉素慢性衰减 mTOR 可以改善老年大鼠学习记忆障碍,阻止神经血管解偶联,恢复脑灌注。此外,对海马和皮层的形态和生化分析表明,mTOR 在老化过程中导致与年龄相关的突触和血管密度下降。这些数据表明,除了介导 AD 和动脉粥样硬化模型中类似 AD 的认知和脑血管缺陷外,mTOR 还促进与正常老化相关的脑血管、神经元和认知缺陷。因此,mTOR 抑制剂可能具有治疗与年龄相关的脑血管功能障碍和认知能力下降的潜力。由于治疗老年人与年龄相关的脑血管功能障碍有望防止脑灌注的进一步恶化,mTOR 衰减可能阻断 AD 的发生和发展。最近,mTOR 被确定为 AD 早期(临床前)阶段的生物标志物。

1 INTRODUCTION

1引言

Normal brain aging predisposes vulnerable neurons to degeneration and is associated with cognitive decline and an increased likelihood of developing a neurodegenerative disorder (Mattson & Magnus, 2006). The prevalence of Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is expected to double approximately every 20 years, with 131.5 million cases expected worldwide by 2050 (Prince et al., 2015). The prevalence of AD is increasing rapidly, yet there is no disease‐modifying treatment currently available. Although age is a primary risk factor for AD, very little is known about the molecular mechanisms that link the regulation of brain aging to neurodegenerative diseases of advanced age.

正常的大脑老化使脆弱的神经元易于退化,并且与认知能力下降和神经退行性紊乱的可能性增加有关(Mattson & Magnus,2006)。阿尔茨海默氏病(AD)是老年痴呆症最常见的病因,预计到2050年全球病例将达到1.315亿,大约每20年翻一番。阿尔茨海默病的患病率正在迅速增加,但目前还没有可用的疾病修正治疗。虽然年龄是 AD 的主要危险因素,但是对于大脑老化与高龄神经退行性疾病之间的分子机制却知之甚少。

Cerebrovascular dysfunction is a universal feature of aging (Zlokovic, 2011) that includes impaired endothelium‐dependent vasodilation and global and regional decreases in cerebral blood flow (CBF) (Martin, Friston, Colebatch, & Frackowiak, 1991). While decreased CBF does not indicate a particular disease state, reduced CBF is associated with impaired cognitive function (Wang et al., 2016) and decreased neuronal plasticity (Hamadate et al., 2011). Using functional magnetic resonance imaging (fMRI), which measures changes in CBF to infer neuronal activation, it is apparent that the human brain can reorganize and redistribute functional networks to compensate for nonpathologic age‐related impairments. In general, task‐related neural activation becomes more diffuse with advancing age as other brain regions are recruited to maintain task proficiency. However, when cognitive demand exceeds these compensatory mechanisms, performance becomes impaired (Cabeza et al., 1997). Because cerebrovascular dysfunction plays a critical role in the pathogenesis of age‐related neurodegenerative disorders, including AD (Csiszar et al., 2017; Lin et al., 20172013; Van Skike et al., 2018), and manifests early in disease progression (Iturria‐Medina, Sotero, Toussaint, Mateos‐Perez, & Evans, 2016), it is important to investigate the mechanisms underlying cognitive decline and brain vascular deterioration driven by nonpathologic aging.

脑血管功能障碍是衰老的一个普遍特征(Zlokovic,2011) ,包括内皮依赖性血管舒张功能受损和脑血流量(CBF)的全球和区域性减少(Martin,弗里斯顿,Colebatch,& Frackowiak,1991)。虽然 CBF 的降低并不意味着某种特定的疾病状态,但 CBF 的降低与认知功能受损(Wang et al. ,2016)和突触可塑性的降低(Hamadate et al. ,2011)有关。使用功能性磁共振成像功能磁共振成像(fMRI) ,测量脑血流量的变化来推断神经元的激活,很明显,人类大脑可以重组和重新分配功能网络,以补偿非病理性的年龄相关的损伤。一般来说,随着年龄的增长,任务相关的神经活动变得更加分散,因为其他脑区被招募来保持任务熟练程度。然而,当认知需求超过这些补偿机制时,表现就会受损(Cabeza 等人,1997)。因为脑血管功能障碍在与年龄相关的神经退行性疾病的发病机制中扮演着关键的角色,包括 AD (cszar 等人,2017; Lin 等人,2017,2013; Van Skike 等人,2018) ,并且在疾病进展的早期表现(Iturria-Medina,Sotero,Toussaint,Mateos-Perez,& Evans,2016) ,所以研究非病理性老化导致的潜在认知能力下降和脑血管恶化的机制非常重要。

The mammalian/mechanistic target of rapamycin (mTOR) pathway regulates aging in mammals (Wilkinson et al., 2012). mTOR is also expressed throughout the brain, where it is linked to synaptic plasticity and learning and memory (Tang et al., 2002) through the regulation of protein synthesis (Tang et al., 2002) and autophagy (Mizushima, Levine, Cuervo, & Klionsky, 2008). In the aging brain, autophagy is reduced, contributing to the accumulation of aggregated proteins and neurodegeneration (Komatsu et al., 2006). Thus, dysregulation of the mTOR pathway during nonpathologic aging may contribute to cognitive decline, cerebrovascular dysfunction, and a predisposition toward developing neurodegenerative diseases associated with advanced age.

哺乳动物雷帕霉素(mTOR)通路的哺乳动物/机械目标调节老化(威尔金森等人,2012)。mTOR 也在整个大脑中表达,它通过调节蛋白质合成(Tang et al. ,2002)和自噬(Mizushima,Levine,奎尔沃,Klionsky,2008)与突触可塑性和学习和记忆有关。在老化的大脑中,自噬减少,促进聚集蛋白质和神经退行性疾病的积累(小松等人,2006)。因此,非病理性衰老过程中 mTOR 通路的失调可能导致认知能力下降,脑血管功能障碍,以及与高龄相关的神经退行性疾病的易感性。

We have previously shown that mTOR inhibition attenuates cognitive dysfunction in aged mice (Halloran et al., 2012). We and others have also shown that chronic mTOR attenuation with rapamycin can prevent and reverse cognitive and cerebrovascular deficits in several independent mouse models of AD (Lin et al., 20172013;Van Skike et al., 2018) and vascular cognitive impairment (Jahrling et al., 2018), leading to improved cerebrovascular function and preserved cognitive outcomes in these models of age‐related disease. The contribution of mTOR to cerebrovascular deficits is associated with normative aging, though its impact on cognitive outcomes remains unknown. The goal of this study was to test the hypothesis that mTOR drives cerebrovascular and synaptic dysfunction during aging and that chronic mTOR inhibition with rapamycin mitigates nonpathologic age‐related deterioration of cognition and cerebrovascular function in aged rats without underlying disease.

我们先前已经证明 mTOR 抑制能够减轻老龄小鼠的认知功能障碍(Halloran et al. ,2012)。我们和其他人也已经证明,雷帕霉素的慢性 mTOR 衰减可以预防和扭转 AD (Lin 等人,2017年,2013年; Van Skike 等人,2018年)和血管认知障碍(Jahrling 等人,2018年)的几个独立小鼠模型的认知和脑血管缺陷,导致改善脑血管功能和保存这些年龄相关疾病模型的认知结果。mTOR 对脑血管功能缺陷的作用与正常老化有关,但其对认知结果的影响尚不清楚。本研究的目的是验证 mTOR 在衰老过程中引起脑血管和突触功能障碍的假设,以及雷帕霉素慢性抑制 mTOR 可以缓解非病理性年龄相关性衰老大鼠的认知和脑血管功能恶化。

2 RESULTS

2结果

2.1 Age‐related decline in hippocampal‐dependent learning and memory is driven by mTOR

2.1年龄相关的海马依赖性学习记忆能力下降是由 mTOR 引起的

To determine the contribution of mTOR to cognitive deficits during normative aging, we used the Morris water maze to measure hippocampal‐dependent spatial learning and memory in adult (16 months old) and aged (34 months old) rats that were either fed either a diet with empty microcapsules or a diet containing microencapsulated rapamycin at 14 parts per million (ppm) for 15 months starting at 19 months of age. Consistent with prior studies (Novier, Van Skike, Diaz‐Granados, Mittleman, & Matthews, 2013), we found that adult rats swam significantly faster than aged rats, regardless of treatment condition (Figure 1a). Thus, we used path length as measure of performance during training in the MWM since this measure is not impacted by swim speed. Total distance swam declined progressively throughout the 4 days of training (Figure 1b), indicating effective spatial learning among the groups. However, 34‐month‐old aged rats had significantly longer path lengths than the 16‐month‐old adult rats on training days 3 and 4. No significant differences in path length were observed in aged rats treated with rapamycin as compared to adult rats on any training day. Additionally, there were no differences among the groups in the amount of slow swimming below 0.10 m/s during acquisition (Figure 1c).

为了确定 mTOR 在标准衰老过程中对认知缺陷的贡献,我们使用 Morris水迷宫任务测量了成年(16个月)和老年(34个月)大鼠的海马依赖性空间学习和记忆。与之前的研究(Novier,Van Skike,Diaz-Granados,Mittleman,& Matthews,2013)一致,我们发现无论治疗条件如何,成年老鼠的游泳速度明显快于老年老鼠(图1a)。因此,我们使用路径长度作为训练期间的表现测量,因为这种测量不受游泳速度的影响。总游泳距离在整个训练的4天中逐渐下降(图1b) ,表明各组之间有效的空间学习。然而,34个月龄大鼠在训练第3天和第4天的路径长度明显长于16个月龄大鼠。老龄大鼠经雷帕霉素处理后,与成年大鼠相比,在任何训练日均无显著性差异。此外,各组之间在获得过程中,慢游速度低于0.10 m/s 的数量没有差异(图1c)。

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Figure 1 图1Open in figure viewer 打开图形查看器PowerPoint 简报Age‐associated cognitive decline in rats is driven by mTOR. (a) Thirty‐four‐month old aged rats, regardless of treatment condition, displayed slower swim speed compared with 16‐month‐old adults (for each training day, q(12)<4.60, * 老鼠年龄相关的认知能力下降是由 mTOR 驱动的。(a)34个月龄的老年大鼠,不论治疗情况如何,与16个月龄的成年大鼠相比,游泳速度较慢(每训练日,q (12) < 4.60,*p < .018; ** indicates < . 018; * * 表示p < .01). Tukey’s < . 01)post hoc 事后的 tests were applied to a significant main effect of group, 实验组采用显著性主效应实验,F(2,36)=8.96, (2,36) = 8.96,p = .0007 in two‐way repeated measures ANOVA analyses. (b) Aged rats exhibit spatial learning and memory impairments compared with adults ( = . 0007在双向重复测量方差分析分析。(b)与成年大鼠相比,老年大鼠表现出空间学习和记忆障碍(F(2, 36)=5.40, (2,36) = 5.40,p = .009), especially on days 3 (**q(144)=4.23, = 0.009) ,特别是在第3天(* * q (144) = 4.23,p = .009) and 4 (*q(144)=3.35, = . 009)及4(* q (144) = 3.35,p = .049) of training in the Morris water maze (MWM). Performance of aged rats in which mTOR was chronically attenuated with rapamycin (aged + rapa) did not significantly differ from that of adult rats for each training day (q(144)<2.56, = 0.049)的培训 Morris水迷宫任务。老龄大鼠经雷帕霉素(rapa)慢性减毒 mTOR 后,每天的运动量与成年大鼠相比无显著性差异(q (144) < 2.56,p < .17, n.s.). Tukey’s < . 17,n.s.)post hoc事后的 tests were applied to significant main effects of day ( 试验采用显著性主效应分析方法,研究了不同剂量(100、100、100、100、100、100、100、100、100、100、F(3,108) = 48.59, p < .0001) and group ( < . 0001)及F(2,36)=5.40, (2,36) = 5.40,p = .009) in two‐way ANOVA (day x group) with repeated measures analyses. (c) The proportion of swimming slower than 0.10 m/s decreases with training ( = 0.009)进行双因素方差分析(第 x 组)及重复测量分析。(c)游泳速度低于0.10米/秒的比例随训练而下降(F(3,108)=12.87, (3,108) = 12.87,p < .0001), but is not different among groups ( < . 0001) ,但不同组别之间没有差异(F(2, 36)=1.93, (2,36) = 1.93,p = .16). (d) Aged rats exhibit significant spatial memory impairment in the probe trial compared with adult rats (*q(144)=3.81,与成年大鼠相比,老年大鼠空间记忆显著减退(* q (144) = 3.81,p = .021). Inhibition of mTOR restores spatial memory in aged rats (aged + rapa group) to a level indistinguishable from that of adult animals (q(144)=1.17,= 0.021).mTOR 的抑制作用使老龄大鼠(+ rapa 组)的空间记忆恢复到与成年大鼠无差别的水平(q (144) = 1.17,p = .69). Tukey’s post hoc tests were applied to a significant main effect of group ( 方法: 采用 Tukey 的事后特别测验,对照组和对照组均有显著的主效应(pF(3, 108)=24.86 (3,108) = 24.86p < .0001) via two‐way ANOVA. Data are presented as mean ±  < . 0001) ,数据以平均值表示SEM 扫描电镜 (n = 10‐15/group) = 10-15/组)

During a 24‐hr probe trial, aged rats made significantly fewer passes over the learned location of the hidden platform as compared to adult rats during a 24‐hr recall probe trial (Figure 1d). Recall of the hidden platform location in aged rats treated with rapamycin, however, was indistinguishable from that of adult rats. Together, these data indicate that deficits in spatial learning and memory in aged rats can be negated by mTOR attenuation, suggesting that spatial learning and memory impairments in aged rats are at least partially driven by mTOR. Of note, chronic mTOR inhibition did not rescue age‐related decreases in swim speed, ruling out an impact of mTOR attenuation on neuromotor pathways or muscle function and activity required for swimming.

在一个24小时的探针试验中,在一个24小时的回忆探针试验中,与成年老鼠相比,老年老鼠在隐藏平台的习得位置上传递的次数明显减少(图1d)。然而,在雷帕霉素治疗的老年大鼠中,回忆起隐藏平台的位置与成年大鼠没有区别。总之,这些数据表明老年大鼠的空间学习和记忆缺陷可以被 mTOR 衰减所抵消,这表明老年大鼠的空间学习和记忆损伤至少部分是由 mTOR 驱动的。值得注意的是,慢性 mTOR 抑制并不能挽救与年龄相关的游泳速度下降,排除了 mTOR 衰减对神经运动通路或肌肉功能和游泳所需活动的影响。

2.2 mTOR drives sensory‐evoked functional hyperemia impairments in rats of advanced age

2.2 mTOR 驱动高龄大鼠感觉诱发的功能性充血损害

Optimal brain function depends on regulation of CBF in response to neuronal activity, through a complex mechanism known as neurovascular coupling (NVC). Decreased NVC occurs during aging in both humans (Fabiani et al., 2014) and rodents (Toth et al., 2014). Functional MRI (fMRI) measures the hemodynamic response of NVC in response to a defined stimulus as an indicator of neuronal activation. Since decreased neuronal activation and impaired cognitive performance during aging have been linked in humans (Cabeza et al., 1997), we used fMRI to determine overall neuronal activity and neurovascular coupling in our rat model of advanced age measured as the hemodynamic response associated with neuronal activation in response to somatosensory (forepaw) stimulation. We found that evoked fMRI response to somatosensory stimulation was blunted in aged rats as compared to adult animals. Chronic mTOR attenuation by rapamycin, however, restored the fMRI response in aged rats to levels indistinguishable from those of adult animals (Figure 2). These results indicate that mTOR attenuation can restore profound deficits in neurovascular coupling responses in aged rats, suggesting that deficient neuronal network activation and/or impaired functional hyperemia during aging are mediated by mTOR.

最佳的大脑功能依赖于调节脑血流对神经元活动的反应,通过一个复杂的机制称为神经血管耦合(NVC)。在人类的衰老过程中,NVC 下降(Fabiani 等人,2014年)和啮齿类动物(Toth 等人,2014年)。功能性磁共振成像(fMRI)测量的血流动力学反应 NVC 的特定刺激作为一个指标的神经元活化。由于在人类衰老过程中神经元激活减少和认知能力受损有关(Cabeza et al. ,1997) ,我们使用功能磁共振成像来确定整体神经元活动和神经血管耦合在我们的高龄大鼠模型中测量的血流动力学反应与神经元激活有关的躯体感觉(前爪)刺激。我们发现,与成年动物相比,老年大鼠对体感刺激的诱发 fMRI 反应变迟钝。然而,雷帕霉素的慢性 mTOR 衰减使老年大鼠的 fMRI 反应恢复到与成年大鼠无法区分的水平(图2)。这些结果表明,mTOR 衰减可以恢复老龄大鼠神经血管耦联反应的严重缺陷,提示 mTOR 参与了衰老过程中神经元网络的激活和/或功能性充血的受损。

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Figure 2 图2Open in figure viewer 打开图形查看器PowerPoint 简报mTOR drives impaired neuronal network activation during aging in rats. (a) Representative fMRI activation in the somatosensory cortex and (b) quantitative analysis demonstrates the response to forepaw stimulation is decreased with age (*** indicates 老龄大鼠 mTOR 对神经元网络活化的影响。(a)躯体感觉皮层的功能磁共振成像(fMRI)和(b)定量分析表明,对前爪刺激的反应随着年龄的增长而减弱(* * *p < .001 via < 0.001经t test). fMRI activation, however, is preserved in aged rats treated with rapamycin (***, 然而,老龄大鼠经雷帕霉素(* * * ,p < .001 compared with age‐matched controls). The restoration of fMRI activation in response to forepaw stimulation by mTOR attenuation was complete since the magnitude of the evoked response in the rapamycin‐treated aged group was indistinguishable from that of adult rats. Evoked responses are shown as mean percent increase over baseline cerebral blood flow ±  < . 001).由于雷帕霉素治疗老年组的诱发反应幅度与成年组大鼠无明显差异,因此雷帕霉素衰减对前爪刺激的功能磁共振成像反应完全恢复。诱发反应显示为比基线脑血流量平均增加%SEM 扫描电镜

2.3 mTOR contributes to decreased presynaptic density with age

2.3 mTOR 参与了突触前密度的降低

Since the decrease in functional hyperemia during somatosensory stimulation (i.e., neurovascular coupling) is dependent on the integrity of both neuronal and vascular responses, we quantified presynaptic density to provide a measure of neuronal integrity. Decreased presynaptic density is associated with mild cognitive impairment and AD (Scheff et al., 2015) and with cognitive impairment in rodents (Wang et al., 2014). To define whether changes in synaptic integrity occur during normative aging in rats and understand the role of mTOR, we measured presynaptic density in rats after completion of training and testing in the Morris water maze. Density (Figure 3a and b) and quantity (Figure 3a and c) of synaptophysin‐positive synaptic boutons in hippocampal CA1 were decreased with advanced age in rats. Both density and quantity of synaptophysin‐positive synaptic elements in aged rats treated with rapamycin to attenuate mTOR, however, were indistinguishable from those of adult animals (Figure 3a–c). Together, these findings indicate that chronic mTOR attenuation curtails an age‐related loss of synaptic boutons in the hippocampus, suggesting that preserved presynaptic integrity by mTOR attenuation may underlie the restoration of hippocampal‐dependent learning and memory and the maintenance of fMRI responses to somatosensory stimulation in aged rats. These data suggest that mTOR dysregulation drives age‐related structural remodeling of the hippocampus during aging in the rat and that mTOR attenuation may block age‐related impairments in hippocampal‐dependent memory through the preservation of presynaptic density.

由于在体感刺激过程中功能性充血的减少(即神经血管耦联)依赖于神经元和血管反应的完整性,我们量化突触前密度来提供神经元完整性的测量。降低突触前密度与轻微认知障碍和 AD 有关(Scheff et al. ,2015) ,并与啮齿类动物的认知障碍有关(Wang et al. ,2014)。为了确定突触完整性的变化是否发生在大鼠的正常老化过程中,并且理解 mTOR 的作用,我们在大鼠完成训练和测试之后,测量了大鼠的突触前 Morris水迷宫任务。大鼠海马 CA1区突触素阳性突触束扣的密度(图3a 和 b)和数量(图3a 和 c)随年龄增长而减少。然而,雷帕霉素衰减 mTOR 老龄大鼠突触素阳性突触元件的密度和数量与成年动物无法区分(图3a-c)。综上所述,这些发现表明 mTOR 的慢性衰减减少了与年龄相关的海马突触结构的丢失,提示 mTOR 衰减保持突触前完整性可能是老年大鼠海马依赖性学习和记忆恢复和维持对躯体感觉刺激的 fMRI 反应的基础。这些数据表明,mTOR 调节异常促使老龄大鼠海马的年龄相关结构重塑,mTOR 衰减可能通过保持突触前密度而阻断海马依赖性记忆的年龄相关性损伤。

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Figure 3 图3Open in figure viewer 打开图形查看器PowerPoint 简报mTOR‐dependent deterioration of presynaptic density during aging. (a) Representative images of synaptophysin immunofluorescent reactivity in hippocampal CA1. (b) Decreased synaptophysin density in aged as compared to adult rats (**, q(9)=7.25, 老化过程中 mTOR 依赖的突触前密度恶化。(a)海马 CA1区突触素免疫荧光反应的典型图像。(b)老年大鼠突触素密度低于成年大鼠(* * ,q (9) = 7.25,p = .002) was significantly ameliorated by chronic mTOR attenuation using rapamycin in the aged + rapa treatment group (*q(9)= 4.31,老年 + 雷帕霉素治疗组经雷帕霉素慢性衰减 mTOR (* q (9) = 4.31,p = .03 vs. aged + vehicle). Synaptophysin density was restored to levels not significantly different from those of adult rats (q(9)=2.93, = 0.03 vs. 年龄 + 车辆)。突触素密度恢复到与成年大鼠无显著差异的水平(q (9) = 2.93,p = .15) in the aged + rapa treatment group. Tukey’s = . 15)老年 + 拉帕治疗组post hoc 事后的 tests were applied to a significant omnibus one‐way ANOVA,本研究采用单因素方差分析法进行统计分析,F(2,9)=13.29, (2,9) = 13.29,p = .002). Data are mean ±  = 0.002)。数据是平均值SEM 扫描电镜 of 的n = 4. (c) Aged rats have fewer synapses as shown by decreased synaptophysin reactive area (****q(9)=13.41, = 4. (c)老龄大鼠突触反应面积减少(* * * q (9) = 13.41,p < .0001), a difference that was abolished by mTOR attenuation in aged rats treated with rapamycin (q(9)=12.57, 雷帕霉素(q (9) = 12.57,p < .0001). Tukey’s < . 0001)post hoc 事后的 tests were applied to a significant one‐way ANOVA ( 方差分析显著性单因素方差分析(F(2,9)=56.44, (2,9) = 56.44,p < .0001). Data are presented as mean ±  < . 0001)。数据以平均值表示SEM 扫描电镜 of 的n = 4 = 4

2.4 Attenuation of mTOR restores cerebral blood flow and sensory‐evoked functional hyperemia in aged rats

2.4衰减 mTOR 恢复老龄大鼠脑血流和感觉诱发功能性充血

Decreased brain perfusion during normative aging has been established (Melamed, Lavy, Bentin, Cooper, & Rinot, 1980), and we recently showed that mTOR mediates cerebrovascular dysfunction in two different mouse models of AD (Lin et al., 20172013) and in a model of cognitive impairment associated with atherosclerosis (Jahrling et al., 2018). To define the role of cerebrovascular dysfunction in impaired sensory‐evoked functional hyperemia in aged rats (Figure 2), we used arterial spin labeling MRI to measure global and regional resting CBF in adult and aged rat groups (Figure 4). Aged rats had significant impairments in global, cortical, and hippocampal resting CBF as compared to adult animals (Figure 4b‐d), and mTOR attenuation with rapamycin restored global, cortical, and hippocampal resting CBF in aged rats (Figure 4b‐d). These data indicate that mTOR activity drives cerebral hypoperfusion during normative aging.

在标准衰老过程中减少的脑灌注已经建立(Melamed,Lavy,Bentin,Cooper,& Rinot,1980) ,我们最近表明 mTOR 介导了两个不同的 AD 小鼠模型(Lin 等人,2017,2013)和一个与动脉粥样硬化相关的认知损害模型中的脑血管功能障碍(Jahrling 等人,2018)。为了明确脑血管功能障碍在老年大鼠感觉诱发功能性充血受损中的作用(图2) ,我们使用动脉自旋标记 MRI 测量成年和老年大鼠组的全局和局部静息脑血流量(图4)。与成年动物相比,老年大鼠在全脑、皮层和海马休息时的脑血流量有明显的损伤(图4b-d) ,雷帕霉素减少 mTOR,恢复了老年大鼠的全脑、皮层和海马休息时的脑血流量(图4b-d)。这些数据表明,mTOR 活动促使正常老化期间脑灌注不足。

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Figure 4 图4Open in figure viewer 打开图形查看器PowerPoint 简报mTOR drives global and regional cerebral hypoperfusion in aging. (a) Representative images of resting CBF obtained by arterial spin labeled MRI. (b) Age‐related cerebral hypoperfusion, evidenced by decreased global CBF (** mTOR 在老化过程中促使全脑和局部脑灌注不足。(a)动脉旋转标记 MRI 获取静息时脑血流的代表性图像。(b)年龄相关的大脑低灌注,证明全脑血流量减少(* *p < .01) in aged rats, is restored by chronic mTOR attenuation in the aged + rapa group (** 目的: 探讨衰老大鼠 mTOR 的衰减规律及其对衰老大鼠 mTOR 的影响p < .01 compared with aged control). (c) Cortical CBF is reduced in aged animals compared with adults (* (c)老龄动物脑皮质血流量低于成年动物(*p < .05), but restored to levels indistinguishable from those of adult rats by chronic mTOR inhibition in the aged + rapa group (* < . 05) ,但老年 + 复发组的 mTOR 慢性抑制恢复到与成年大鼠无差别的水平(*p < .05 compared with aged animals). (d) Reduced hippocampal CBF in aged rats as compared to adults (* (d)老龄大鼠海马脑血流量低于成年大鼠(*p < .05) is abrogated with chronic mTOR attenuation in the aged + rapa group (* < . 05)随着老年 + 复发组 mTOR 的慢性衰减而消失(*p < .05 compared with aged rats) as a result of Student’s (与老年大鼠比较 < . 05)t test 测试

2.5 mTOR attenuation restores cortical microvascular density in aged rats

2.5 mTOR 衰减恢复老龄大鼠皮层微血管密度

Because impaired CBF and blunted functional hyperemia responses could arise from decreased cerebral microvascular density, we assessed cortical and hippocampal microvascular density directly using immunofluorescence in tissue to label microvascular endothelial cells in combination with confocal microscopy and quantitative measures of endothelial cell reactivity on serial sections through parietal cortex and hippocampal CA1. Aged rats showed significantly reduced cortical microvascular density in those brain regions as compared to adult animals (Figure 5a–b). Cortical microvascular density in aged rats treated with rapamycin, however, was indistinguishable from that of adult rats (Figure 5a–b). Similar to cortex, hippocampal microvascular density was significantly decreased in aged rats compared with adults (Figure 5c–d). Attenuation of mTOR, however, restored microvascular density in rapamycin‐treated aged rats to levels indistinguishable from those of adult animals (Figure 5c–d). Taken together, these data indicate that mTOR drives microvascular density loss in cortex and hippocampus during normative aging in rats and implicates mTOR‐dependent microvascular rarefaction in the etiology of decreased CBF and impaired functional hyperemia during aging in rats.

由于 CBF 受损和功能性充血反应迟钝可能是由于脑微血管密度降低引起的,我们直接利用组织中的免疫荧光标记微血管内皮细胞,结合共聚焦显微镜和内皮细胞反应性的定量测量,在穿过顶叶皮层和海马 CA1区的连续切片上进行测量,评估皮层和海马微血管密度。与成年大鼠相比,老年大鼠大脑这些区域的皮质微血管密度显著降低(图5a-b)。然而,雷帕霉素治疗的老龄大鼠皮层微血管密度与成年大鼠无法区分(图5a-b)。与皮层相似,老龄大鼠海马微血管密度较成年大鼠显著降低(图5c-d)。然而,mTOR 的衰减使雷帕霉素治疗的老年大鼠微血管密度恢复到与成年大鼠无法区分的水平(图5c-d)。综上所述,这些数据表明 mTOR 在大鼠正常衰老过程中促进了皮质和海马微血管密度的丢失,提示 mTOR 依赖的微血管稀疏是大鼠衰老过程中 CBF 降低和功能性充血障碍的病因。

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Figure 5 图5Open in figure viewer 打开图形查看器PowerPoint 简报mTOR contributes to age‐related loss of microvascular density in cortex and hippocampus. Representative images of (a) cortical and (b) hippocampal microvasculature highlighted with Alexa488‐tomato lectin labeling of endothelial cells. Quantitative analyses demonstrate decreased microvascular density in (c) cortex of aged rats compared with adult rats (*q(54)=3.48, mTOR 参与了年龄相关的皮质和海马微血管密度的损失。用 Alexa488-tomato 凝集素标记内皮细胞,突出显示(a)皮层和(b)海马微血管的典型图像。定量分析显示老龄大鼠皮层微血管密度较成年大鼠(* q (54) = 3.48,p = .045), which is negated by chronic mTOR attenuation in the aged + rapa group (compared with aged, *q(54)=4.12, 老年 + 复发组 mTOR 慢性衰减(与老年组比较 * q (54) = 4.12,p = .014); and in (d) hippocampus of aged rats compared with adults (*q(27)=4.19, = . 014) ; (d)老年大鼠与成年大鼠的海马(* q (27) = 4.19,p = .017), which is attenuated with chronic mTOR inhibition in the aged + rapa group (vs. adult, q(27)=1.99, 老年 + 复发组(与成年组比较,q (27) = 1.99,p = .35). Data from cortex (a‐b) are from 3 independent fields in 6–7 rats per group for a total = 0.35).皮层(a-b)数据来源于每组6-7只大鼠的3个独立电场n = 18–21 in the analysis; data from hippocampus (c‐d) are from 2 independent fields in 4–6 animals per group for a total of = 18-21在分析中; 数据来自海马(c-d)2个独立的领域,每组4-6动物,共计n = 8–12 in the analysis. Data are displayed as mean ±  = 8-12,数据显示为平均值 ±SEM 扫描电镜 normalized to the adult control group for all studies 所有研究都归为成人对照组

3 DISCUSSION

3讨论

Increased age is the greatest risk factor for AD (Guerreiro & Bras, 2015). Impaired cerebrovascular function during aging (Hamadate et al., 2011;Martin et al., 1991;Wang et al., 2016) is, in turn, a biomarker for increased risk of AD (Zlokovic, 2011) and is one of the earliest detectable changes in the disease pathogenesis (Iturria‐Medina et al., 2016). Consistent with prior reports showing that mTOR inhibition improves learning and memory in aged mice (Halloran et al., 2012;Majumder et al., 2012), our data indicate that chronic mTOR inhibition reduces age‐dependent impairments in spatial learning and memory and that the improved cognitive outcomes are associated with the preservation of synaptic integrity (Figure 3), neuronal network activation (Figure 2), microvascular integrity (Figure 5), and cerebrovascular function (Figure 4) during aging.

年龄增长是 AD 的最大危险因素(Guerreiro & Bras,2015)。在衰老过程中受损的脑血管功能(Hamadate 等人,2011年; Martin 等人,1991年; Wang 等人,2016年)反过来是 AD 风险增加的生物标志物(Zlokovic,2011年) ,是疾病发病机制中最早可检测到的变化之一(Iturria-Medina 等人,2016年)。与之前的研究表明 mTOR 抑制能够提高老年小鼠的学习和记忆能力的报告一致(Halloran 等人,2012; Majumder 等人,2012) ,我们的数据表明,慢性 mTOR 抑制能够减少空间学习和记忆方面的年龄依赖性损伤,并且认知结果的改善与老年期突触完整性的保存有关(图3) ,神经元网络的激活(图2) ,微血管完整性(图5)和脑血管功能(图4)。

Presynaptic synaptophysin expression decreases naturally with nonpathologic aging (Tucsek et al., 2017). Further, lack of functional synaptic protein expression, including synaptophysin, is associated with hippocampal‐dependent memory impairment (Schmitt, Tanimoto, Seeliger, Schaeffel, & Leube, 2009). Consistent with these data, we found that mTOR activity decreased synaptophysin quantity and density (Figure 3) in the hippocampus, suggesting that age‐related synaptic loss may underlie impairments in neuronal network activation (Figure 2) and may contribute to spatial learning and memory deficits (Figure 1) in aged rats. Although mTOR is essential for synaptic function, there is a critical level of mTOR activity that produces optimal synaptic function. For instance, near complete inhibition of mTOR activation during LTP with pharmacogenetics (Stoica et al., 2011) and hyperactivation of mTOR arising from functional loss of negative regulators of the kinase (Lugo et al., 2014) are both detrimental to neuronal function. In contrast, studies have demonstrated that moderate reduction of mTOR activity by ~25%–30% consistently improves aspects of brain function in models of aging (Halloran et al., 2012) and of age‐associated neurological disease (Jahrling et al., 2018; Lin et al., 20172013; Van Skike et al., 2018). Additionally, the mild effects of mTOR inhibition on neuronal function may be a consequence of the relatively poor blood–brain barrier permeability of these compounds (~12,500 fold lower in brain relative to blood, as reported in the methods section). These data suggest the relationship between mTOR activity and cognitive function follows an inverted U‐shaped dose‐effect curve, where very low and very high levels of mTOR activity are deleterious, and enhancement of outcomes (or restoration of disease‐induced deficits) is observed at moderate levels of mTOR, with maximum functional performance occurring at levels of mTOR activity that are reduced by 25%–30% relative to control baseline.

突触前突触素的表达随着非病理性老化自然减少(tucket al. ,2017)。此外,缺乏功能性突触蛋白表达,包括突触素,与海马依赖性记忆损害有关(Schmitt,Tanimoto,Seeliger,Schaeffel,& Leube,2009)。与这些数据一致,我们发现 mTOR 活性降低了海马突触素的数量和密度(图3) ,这表明与年龄相关的突触丢失可能是神经元网络活动受损的原因(图2) ,并可能导致老年大鼠的空间学习和记忆缺陷(图1)。虽然 mTOR 对于突触功能是必不可少的,但是 mTOR 活动的临界水平可以产生最佳的突触功能。例如,在 LTP 过程中使用遗传药理学抑制 mTOR 激活接近完全,以及由于失去负性激酶调节因子而导致 mTOR 过度激活都对神经元功能有害。相比之下,研究表明,适度降低 mTOR 活性约25%-30% ,在衰老模型(Halloran 等人,2012年)和年龄相关的神经系统疾病模型(Jahrling 等人,2018年; Lin 等人,2017年,2013年; Van Skike 等人,2018年)中持续改善大脑功能的各个方面。此外,mTOR 抑制对神经元功能的轻度影响可能是这些化合物血脑屏障通透性相对较差的结果(如方法部分所报道的,相对于血液,大脑的通透性要低12,500倍)。这些数据表明,mTOR 活动与认知功能之间的关系遵循一个倒 u 型剂量效应曲线,其中 mTOR 活动水平非常低和非常高是有害的,在中度 mTOR 水平上观察到结果的改善(或恢复疾病引起的缺陷) ,最大功能表现发生在 mTOR 活动水平相对于对照基线减少25%-30% 的水平上。

The regulation of CBF during neuronal network activation is a tightly coupled process that depends on the complex interaction between neurons, astrocytes, and vasculature in a process known as neurovascular coupling (Tarantini et al., 2018). Neurovascular coupling reflects both the neuronal activity and the corresponding hemodynamic response that ensures delivery of critical metabolic substrates, largely glucose and oxygen, to active neuronal networks (Tarantini et al., 2018). Chronic mTOR inhibition prevented age‐related declines in global and regional resting CBF, an early biomarker of brain aging in humans (Martin et al., 1991;Schultz et al., 1999). Age‐related cerebral hypoperfusion could be partially explained by microvascular rarefaction, since we found a profound decrease in microvascular density in cortex and hippocampus of aged rats (Figure 5). Microvascular density loss during aging, however, was negated by chronic mTOR attenuation, suggesting that preservation of microvascular density by mTOR attenuation may underlie the protection of CBF in rapamycin‐treated aged rats. These data strongly implicate mTOR as a driver of microvascular rarefaction and dysfunction during aging, and reveal mTOR attenuation as a potentially useful approach to diminish early age‐related CBF loss (Martin et al., 1991; Schultz et al., 1999).

神经元网络激活过程中 CBF 的调节是一个紧密耦合的过程,依赖于神经元、星形胶质细胞和血管之间的复杂相互作用,这一过程被称为神经血管耦合(Tarantini et al. ,2018)。神经血管耦联反映了神经元的活动和相应的血液动力学反应,这种反应确保了关键的代谢基质(主要是葡萄糖和氧气)传递到活跃的神经元网络(Tarantini 等,2018年)。慢性 mTOR 抑制阻止了全球和区域静息脑血流量的衰退,这是人类大脑老化的早期生物标志物(Martin 等,1991; Schultz 等,1999)。年龄相关的脑灌注不足可以部分地解释为微血管稀疏,因为我们发现老年大鼠大脑皮层和海马的微血管密度明显降低(图5)。衰老过程中微血管密度的损失与 mTOR 的慢性衰减无关,提示 mTOR 衰减对微血管密度的保护作用可能是雷帕霉素对衰老大鼠 CBF 的保护作用的基础。这些数据强烈暗示 mTOR 是老化过程中微血管稀疏和功能障碍的驱动因素,并揭示 mTOR 衰减作为减少早期年龄相关脑血流损失的潜在有用途径(Martin 等人,1991; Schultz 等人,1999)。

The studies discussed above indicate that mTOR directs aspects of both neuronal and microvascular dysfunction and loss during normative aging in rats. Attenuation of evoked somatosensory fMRI signals in aged rats as compared to adults and their restoration in rapamycin‐treated aged animals likely reflects the mTOR‐mediated loss of both neuronal and vascular components that are necessary for neurovascular coupling. Thus, mTOR‐driven neuronal and/or microvascular dysfunction may have initiated neurovascular uncoupling during normative aging in rats. Future cross‐sectional or longitudinal studies will identify this apical mTOR‐dependent injury.

以上讨论的研究表明,mTOR 指导大鼠在正常衰老过程中神经元和微血管功能障碍和丢失的各个方面。与成年大鼠相比,老年大鼠诱发性体感功能磁共振成像信号的衰减及雷帕霉素对老年大鼠的恢复可能反映了 mTOR 介导的神经元和血管组分的丢失,而这些组分是神经血管耦合所必需的。因此,mTOR 驱动的神经元和/或微血管功能障碍可能在大鼠正常衰老过程中引发了神经血管解偶联。未来的横断面或纵向研究将确定这种心尖 mTOR 依赖性损伤。

It has been suggested that decreased cerebral microvascular density during aging (Sonntag, Lynch, Cooney, & Hutchins, 1997) may underlie reduced microvascular plasticity and synaptogenesis in aged rats after environmental enrichment (Black, Polinsky, & Greenough, 1989). Loss of angiogenesis during aging may also contribute to reduced microvascular density and synaptic loss. Alternatively, loss of synaptic integrity with age may subsequently trigger microvascular dysfunction and disintegration, and it is possible that either event may initiate age‐associated cerebrovascular and neuronal dysfunction.

有人提出,衰老过程中脑微血管密度的降低可能是环境丰容后老年大鼠微血管可塑性和突触发生减少的原因(Sonntag,Lynch,Cooney 和 Hutchins,1997)。老化过程中血管生成的缺失也可能导致微血管密度的降低和突触的丢失。另外,随着年龄的增长,突触完整性的丧失可能引起微血管功能障碍和解体,这两种情况都有可能引发年龄相关的脑血管和神经元功能障碍。

The beneficial effects of mTOR inhibition we observed in this study may also be related to factors that were not measured in the present study. For instance, mTOR regulates microglial activation states as well as the secretion of proinflammatory cytokines and chemokines (Li et al., 2016). Therefore, some effects may be due to the downregulation of mTOR‐driven neuroinflammatory responses, which increase during aging and negatively impact both neuronal and brain vascular function. Additionally, the central role of oxidative stress in age‐related cerebrovascular dysfunction (Sure et al., 2018) was recently demonstrated in studies showing that the age‐related deterioration of cerebrovascular endothelial cell function and neurovascular coupling responses were reversed by reducing oxidative stress in endothelial cells (Kiss et al., 2019; Tarantini et al., 20192018). Inhibition of mTOR is protective against oxidative stress in vitro in vascular endothelial cells (Zheng et al., 2017), indicating that mTOR may also regulate oxidative stress. While we have not addressed the role of mTOR‐driven neuroinflammation or oxidative stress in our studies, these may represent important mechanisms through which mTOR inhibition ameliorates cerebrovascular dysfunction and cognitive impairment in the aging brain. Figure 6 illustrates the interaction and potential convergence of mTOR‐driven pathways of brain aging.

我们在这项研究中观察到的 mTOR 抑制的有利影响也可能与本研究中没有测量的因素有关。例如,mTOR 调节小胶质细胞的活化状态以及前炎症细胞因子和趋化因子的分泌(Li et al. ,2016)。因此,一些影响可能是由于 mTOR 驱动的神经炎症反应的下调,这种下调在衰老过程中增加,并对神经元和脑血管功能产生负面影响。此外,氧化应激在年龄相关的脑血管功能障碍中的中心作用(Sure 等人,2018年)最近的研究表明,年龄相关的脑血管内皮细胞功能和神经血管耦联反应的恶化是通过减少内皮细胞的氧化应激来逆转的(Kiss 等人,2019; Tarantini 等人,2019,2018年)。在血管内皮细胞中,mTOR 的抑制对氧化应激有保护作用(Zheng et al. ,2017) ,这表明 mTOR 也可能调节氧化应激。虽然在我们的研究中还没有涉及 mTOR 驱动的神经炎症或氧化应激的作用,但这些可能代表了 mTOR 抑制改善脑血管功能障碍和老化大脑认知功能障碍的重要机制。图6说明了 mTOR 驱动的脑老化通路的相互作用和潜在的趋同性。

image
Figure 6 图6Open in figure viewer 打开图形查看器PowerPoint 简报Proposed mTOR‐dependent mechanisms of brain aging. mTOR inhibitors, including rapamycin, rapalogs, and kinase inhibitors, can restore neuronal and cerebrovascular function by blocking specific mTOR‐dependent pathways of brain aging 提出的 mTOR 依赖性脑老化机制。mTOR 抑制剂,包括雷帕霉素、雷帕霉素和激酶抑制剂,可以通过阻断特异性的 mTOR 依赖性脑衰老通路来恢复神经元和脑血管功能

In summary, we show that mTOR drives cerebrovascular and neuronal dysfunction associated with cognitive decline during normative aging in the rat. In aged rats, chronic mTOR inhibition with rapamycin, an intervention that extends lifespan (Wilkinson et al., 2012), ameliorated mTOR‐dependent decline in learning and memory in aging at least partly through the restoration of cerebrovascular function and synaptic structure. Together with our previous studies, these data indicate that mTOR drives cerebrovascular dysfunction both in normative aging and in age‐associated disease states, including AD and cognitive impairment associated with vascular disease (Halloran et al., 2012; Jahrling et al., 2018; Lin et al., 20172013; Van Skike et al., 2018) and thereby suggests that brain microvascular dysfunction may link aging to an increased risk of AD. Therefore, mTOR attenuation provides a strategy to preserve synaptic and cerebrovascular function during aging and may help to reduce the risk of AD.

综上所述,我们证明 mTOR 在大鼠正常老化过程中驱动脑血管和神经元功能障碍,并与认知功能下降有关。在老年大鼠中,雷帕霉素慢性抑制 mTOR,延长寿命的干预(Wilkinson 等人,2012) ,至少部分通过脑血管功能和突触结构的恢复,改善了老年大鼠学习和记忆中 mTOR 依赖性的下降。与我们以前的研究一起,这些数据表明 mTOR 在正常老化和年龄相关疾病状态下都会引起脑血管功能障碍,包括与血管疾病相关的 AD 和认知障碍(Halloran et al. ,2012; Jahrling et al. ,2018; Lin et al. ,2017,2013; Van Skike et al. ,2018) ,从而表明大脑微血管功能障碍可能与老化有关,增加 AD 的风险。因此,mTOR 衰减可以提供一种在衰老过程中保护突触和脑血管功能的策略,可能有助于降低 AD 的风险。

Although the safety and tolerability of mTOR inhibition in older adults has generally been demonstrated (Kraig et al., 2018; Mannick et al., 2014), a recent clinical trial in healthy adults over 70 years of age reported relatively mild side effects of daily rapamycin administration for at least 8 weeks, including gastrointestinal issues, facial rash, and stomatitis (Kraig et al., 2018). However, the participants taking rapamycin did not have changes in immune function, insulin sensitivity, insulin secretion, or blood glucose concentration (Kraig et al., 2018). Changes in metabolism and immunological suppression represent significant concerns with chronic mTOR inhibition, but preclinical studies suggest side effects can be managed with dosing regimens that may involve intermittent administration (Arriola Apelo, Pumper, Baar, Cummings, & Lamming, 2016; Dumas & Lamming, 2019). Despite the overwhelming preclinical evidence that rapamycin or other inhibitors of the mTOR pathway may slow the progression of AD and several clinical trials that suggest these medications are well tolerated in older adults, there has not yet been a clinical trial testing rapamycin as a therapeutic to delay or slow disease progression in patients with AD (Kaeberlein & Galvan, 2019). Our findings suggest that mTOR may be a therapeutic target to restore cerebrovascular function during normative aging and that mTOR inhibitors may help decrease the risk of developing age‐associated disorders including vascular‐type dementia and AD (Jahrling et al., 2018; Van Skike et al., 2018). Treatment of age‐related cerebrovascular dysfunction in older adults is expected to prevent further deterioration of brain perfusion, recently identified as a biomarker for the very early (preclinical) stages of AD (Iturria‐Medina et al., 2016), and thus potentially blocking disease initiation and/or progression.

虽然 mTOR 抑制剂在老年人中的安全性和耐受性已经得到了普遍的证实(Kraig 等人,2018; Mannick 等人,2014) ,但是最近的一项针对70岁以上健康成年人的临床试验报告说,每日使用雷帕霉素至少8周会产生相对轻微的副作用,包括胃肠道问题、面部皮疹和口腔炎(Kraig 等人,2018)。然而,服用雷帕霉素的参与者在免疫功能、胰岛素敏感性、胰岛素分泌或血糖浓度方面没有变化(Kraig et al. ,2018)。代谢和免疫抑制的变化代表了对慢性 mTOR 抑制的重大关切,但临床前研究表明,可以通过可能涉及间歇性给药的给药方案来控制副作用(Arriola Apelo,Pumper,Baar,Cummings,& Lamming,2016; Dumas & Lamming,2019)。尽管有压倒性的临床前证据表明雷帕霉素或其他 mTOR 通路抑制剂可能延缓 AD 的进展,并且一些临床试验表明这些药物在老年人中耐受性良好,但尚未有临床试验测试雷帕霉素作为延缓或减缓 AD 患者疾病进展的治疗药物(Kaeberlein & Galvan,2019)。我们的研究结果表明,mTOR 可能是在正常老化期间恢复脑血管功能的治疗靶点,mTOR 抑制剂可能有助于降低发展年龄相关性疾病的风险,包括血管型痴呆和 AD (Jahrling 等人,2018; Van Skike 等人,2018)。治疗老年人与年龄有关的脑血管功能障碍有望防止脑灌注的进一步恶化,最近被确定为 AD 早期(临床前)阶段的生物标志物(Iturria-Medina et al. 2016) ,从而可能阻断疾病的发生和/或进展。

4 EXPERIMENTAL PROCEDURES

4个实验步骤

4.1 Animals and treatment conditions

4.1动物和处理条件

Experiments were performed with approval from the University of Texas Health San Antonio Institutional Animal Care and Use Committee (Animal Welfare Assurance Number A3345‐01), which complies with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training, and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for reporting animal experiments. Sixteen‐month‐ and 34‐month‐old male and female F344BNF1 rats were fed chow containing either microencapsulated rapamycin or vehicle diet containing inactive eudragit capsules. Adult rats were fed vehicle diet for 10 months, beginning at 6 months of age until testing at 16 months. Aged rats were fed either vehicle or rapamycin diets beginning at 19 months of age until testing at 34 months. This formed 3 experimental groups: adult vehicle (n = 10), aged vehicle (n = 14), and aged + rapamycin (n = 15). Aged rats were initially placed on diet containing 42 ppm encapsulated rapamycin for 5 months, but were gradually tapered down to 14 ppm over 2 months due to weight loss and mouth sores observed at the higher dose. Rats remained on 14 ppm diet for the last 8 months of treatment. The average level of rapamycin in uncoagulated blood of aged rats treated with 14 ppm microencapsulated rapamycin in chow was 7.3 ng/ml, which is consistent with studies administering the same diet to C57BL/6 mice (Jahrling et al., 2018). Concentrations found in brain tissue were approximately 12,500 fold lower, at 0.585 pg/mg, which is close to the range of 1–3 pg/mg previously reported in C57BL/6J mice administered the same diet (Lin et al., 2013).

实验是在得克萨斯大学圣安东尼奥健康机构动物护理和使用委员会(动物福利保证编号 A3345-01)的批准下进行的,该委员会遵守《动物福利法》、《实验动物护理和使用指南》、《公共卫生服务人道护理和使用实验动物政策》、《美国政府关于在试验、研究和培训中使用脊椎动物的原则》以及《动物研究: 体内实验报告》(arric)报告动物实验的指南。16个月和34个月大的雄性和雌性 F344BNF1大鼠分别喂食含有微囊化雷帕霉素或含有非活性普拉基胶囊的载体饲料的饲料。成年大鼠被喂以汽车饲料10个月,从6个月大开始直到16个月大时测试。从19个月大开始直到34个月大时测试,老龄大鼠分别被喂以载体或雷帕霉素饲料。实验分为3组: 成人车辆(n = 10)、老年车辆(n = 14)、老年 + 雷帕霉素(n = 15)。最初给老年大鼠喂食含42ppm 封装雷帕霉素的饲料5个月,但由于体重下降和口腔溃疡,2个月后逐渐减少到14ppm。在最后8个月的治疗中,老鼠仍然保持14ppm 的饮食。用14ppm 微囊化雷帕霉素治疗的老龄大鼠未凝血血液中雷帕霉素的平均水平为7.3 ng/ml,这与给予 C57BL/6小鼠相同饮食的研究结果一致(Jahrling et al. ,2018)。脑组织中发现的浓度大约低12,500倍,为0.585 pg/mg,这接近于先前报道的 C57BL/6J 小鼠给予同样饮食的1-3 pg/mg 范围(Lin 等人,2013年)。

4.2 Morris water maze

4.2 Morris水迷宫任务

Rats were trained to navigate to a submerged platform using spatial cues. The pool (150 cm in diameter) was maintained at 23 ± 1°C. Rats were trained for 4 days and received 4 trials per day starting from each of the four quadrants. During each trial, the rat was placed into the maze facing the wall and allowed 60 s to find the platform. If the platform was not located within 60 s, the animal was gently guided to the platform. Rats remained on the platform for 5 s after each trial. When all 4 trials were complete, rats were gently dried and returned to their home cages. A tracking system (Ethovision, Noldus) was used to record the distance traveled to reach the platform and swim speed. Separate two‐way (training day × group) repeated measures ANOVAs were used to analyze distance and swim speed, followed by Tukey’s multiple comparison post hoc tests among all means.

老鼠被训练用空间线索导航到水下平台。水池(直径150cm)维持在23 ± 1 ° c。老鼠训练4天,从四个象限开始每天进行4次试验。在每次试验中,老鼠被放入面向墙壁的迷宫中,并让60秒的老鼠找到平台。如果平台位置不在60秒内,那么动物就会被轻轻地引导到平台上。每次试验结束后,大鼠在平台上停留5秒钟。当所有4项试验完成后,老鼠被轻轻地晾干并放回它们的笼子里。一个跟踪系统(Ethovision,Noldus)被用来记录到达平台的距离和游泳速度。采用单独的双向(训练日 × 组)重复测量方法分析距离和游泳速度,然后采用 Tukey 的多重比较后测方法进行比较。

4.3 Functional neuroimaging

4.3功能神经影像

The MRI experiments were performed on a horizontal 7T/30 cm magnet (Bruker Biospec) at the Research Imaging Institute of UT Health San Antonio. Anesthesia was induced with 4.0% isoflurane and then maintained at 1.2% isoflurane and air mixture using a face mask. Heart rate (90–130 bpm), respiration rate, and rectal temperature (37 ± 0.5°C) were continuously monitored. A water bath with circulating water at 45–50°C was used to maintain the body temperature. Heart rate and blood oxygen saturation level were recorded using a MouseOx system (STARR Life Science) and maintained within normal physiological ranges.

磁共振成像实验是在德州大学圣安东尼奥健康成像研究所的一块7t/30cm 水平磁体(Bruker Biospec)上进行的。麻醉用4.0% 异氟醚诱导,然后维持在1.2% 异氟醚和空气混合物使用面罩。连续监测心率(90ー130bpm)、呼吸频率、直肠温度(37 ± 0.5 °c)。使用45-50 °c 循环水浴,以保持体温。使用 MouseOx 系统(STARR Life Science)记录心率和血上静脉血氧饱和度水平,并维持在正常生理范围内。

Quantitative CBF was measured using MRI‐based continuous arterial spin labeling techniques as previously described (Lin, Zhang, Gao, & Watts, 2015). Briefly, a surface coil was placed on top of the head and a labeling coil was placed on the chest, over the heart for continuous arterial spin labeling. The two coils were actively decoupled. Paired, interleaved images were acquired with field of view = 12.8 × 12.8 mm, matrix = 128 × 128, slice thickness = 1 mm, 10 slices, labeling during = 2,100 ms, repetition time = 3,000 ms, and echo time = 20 ms. Continuous arterial spin labeling image analysis employed codes written in Matlab and STIMULATE software (University of Minnesota) to obtain CBF values.

定量的脑血流量是使用磁共振成像为基础的连续动脉自旋标记技术如前所述(林,张,高,& Watts,2015)。简单地说,一个表面线圈放置在头顶上,一个标记线圈放置在胸部,在心脏上连续动脉自旋标记。两个线圈是主动解耦的。采集成对的交错图像,视野 = 12.8 × 12.8 mm,矩阵 = 128 × 128,切片厚度 = 1 mm,10片,标记时间 = 2,100 ms,重复时间 = 3,000 ms,回波时间 = 20 ms,连续动脉自旋标记图像分析采用 Matlab 和 STIMULATE 软件(明尼苏达大学)编写代码获得 CBF 值。

To perform the fMRI experiments, two needle electrodes were inserted under the skin of the right forepaw: one between the first and second digits and the other between the third and fourth digits. These electrodes were then fixed with surgical tape, and the stimulation was confirmed by observing digit twitching. The forepaw was stimulated simultaneously in series at 10 mA, 12 Hz, and 3 ms pulse width. A paradigm of 30 s on and 30 s off was used for forepaw stimulation with five repetitions. Imaging was processed and the relative blood oxygenation level‐dependent (BOLD) changes were calculated using an in‐house Matlab software.

为了进行 fMRI 实验,两个针状电极插入右前爪的皮肤下: 一个在第一和第二个手指之间,另一个在第三和第四个手指之间。然后用外科胶带固定这些电极,并通过观察手指抽搐来确认刺激。前爪同时在10ma,12hz 和3ms 脉冲宽度串联刺激。前爪刺激采用30s on 和30s off 模式,重复5次。图像处理和相对血氧水平依赖(BOLD)的变化计算使用内部 Matlab 软件。

4.4 Tissue collection and preparation

4.4组织收集和准备

Rats were sacrificed by isoflurane overdose, and brains were rapidly removed and divided into two halves along the longitudinal fissure. One half was snap frozen in on dry ice and sectioned into 10 μM slices with a cryostat for immunohistochemical analysis. The cortex from the remaining half was dissected and made into lysates for Western blotting.

用异氟醚过量处死大鼠,迅速取出大脑沿纵裂分成两半。其中一半用干冰冻结,用恒温器切成10μM 薄片进行免疫组织化学分析。剩下一半的皮质被切开,制成裂解物长达一个西方墨点法。

4.5 Immunofluorescent analysis of microvasculature and synaptic density

4.5微血管和突触密度的免疫荧光分析

Slides were fixed in 4% paraformaldehyde, blocked in 5% bovine serum albumin for 1 hr at room temperature, and incubated with either 488‐conjugated tomato lectin (DyLight488 Lycopersicon Esculentum, Vector Laboratories) or synaptophysin primary antibodies (Millipore Sigma, MAB5258) overnight at 4°C. Slides treated with the synaptophysin primary were washed with PBS, Alexa Fluor 488 secondary was applied for 1 hr, then washed. Coverslips were mounted with Prolong Gold antifade mountant with DAPI (ThermoFisher). A secondary‐only control was included (data not shown) to ensure nonspecific background fluorescence was within acceptable limits. Immunofluorescence was visualized using a Zeiss Axiovert 200 m fluorescent microscope with a 100x/1.3 NA Plan‐Neofluar objective and a Zeiss Axiocam MRm camera.

玻片固定在4% 的多聚甲醛中,在室温下用5% 的牛血清白蛋白凝集素封闭1小时,然后与488结合的番茄凝集素(DyLight488 Lycopersicon Esculentum,Vector 实验室)或突触素初级抗体(Millipore Sigma,MAB5258)在4 ° c 过夜培养。用突触素初级处理的载玻片用 PBS 清洗,Alexa Fluor 488次级处理1小时,然后清洗。用 DAPI (ThermoFisher)延长金防褪色底片安装。一个二级控制包括(数据未显示) ,以确保非特异性背景荧光是在可接受的限度内。使用 Zeiss Axiocam MRm 相机和100x/1.3 NA Plan-Neofluar 物镜,使用公共显微镜200m 荧光显微镜观察免疫荧光。

Tomato lectin immunofluorescence was used to determine the endothelial density of the cortical and hippocampal microvasculature, while synaptophysin immunofluorescence was used to determine presynaptic density in the CA1 of the hippocampus. Three sections of the parietal cortex and three sections of the CA1 were imaged in each rat. Mean gray value of tomato lectin and synaptophysin fluorescence, and synaptophysin‐positive area was measured using ImageJ. Measures from the three sections of each rat were averaged and used for analysis. Separate one‐way ANOVAs followed by Tukey’s post hoc comparisons were used to analyze the data.

用番茄凝集素免疫荧光测定皮层和海马微血管的内皮密度,用突触素免疫荧光测定海马 CA1区的突触前密度。每只大鼠的顶叶皮质的三个部分和 CA1的三个部分被成像。用 ImageJ 测定番茄凝集素、突触素荧光灰度值和突触素阳性面积。取每只大鼠的三个部分的测量值平均,并用于分析。单独的单向 ANOVAs 随后 Tukey 的事后比较被用来分析数据。

4.6 Measurement of rapamycin using HPLC‐tandem MS

4.6液相色谱串联质谱法测定雷帕霉素的含量

Quantification of rapamycin in blood and brain tissue was performed using HPLC‐tandem MS as previously reported (Jahrling et al., 2018; Lin et al., 2013). The HPLC system consisted of a Shimadzu SCL‐10 A Controller, LC‐10AD pump with a FCV‐10Al mixing chamber, SIL‐10AD autosampler, and an AB Sciex API 3,200 tandem mass spectrometer with turbo ion spray. The analytical column was a Grace Alltima C18 (4.6–150 mm, 5 m) maintained at 60°C during the chromatographic runs using a Shimadzu CTO‐10A column oven.

血液和脑组织中雷帕霉素的定量使用高效液相色谱-串联质谱以前报道(Jahrling 等人,2018; Lin 等人,2013)。高效液相色谱系统由岛津 SCL-10a 控制器、 FCV-10Al 混合室 LC-10AD 泵、 SIL-10AD 自动采样器和 AB sciexapi3,200涡轮离子喷雾串联质谱仪组成。分析柱是 Grace Alltima C18(4.6-150毫米,5米) ,在 Shimadzu CTO-10A 柱炉进行色谱运行时保持在60 ° c。

Rapamycin was quantified in rat brain according to the following protocol. Briefly, 100 mg of calibrator or brain samples were sonicated with 10 ml of 0.5 mg/ml ascomycin (internal standard) and 300 ml of a solution containing 0.1% formic acid and 10 mM ammonium formate dissolved in 95% HPLC grade methanol. After sonication, the samples were vortexed and centrifuged at 15,000 g for 5 min at 23°C. Supernatants were transferred to 1.5 ml microfilterfuge tubes and spun at 15,000 g for 1 min. 40 ml of the final extracts was injected into the LC/MS/MS. The ratio of the peak area of rapamycin to that of the ascomycin standard (response ratio) for each unknown sample was compared against a linear regression of calibrator response ratios to quantify rapamycin.

雷帕霉素在大鼠脑中按照下列方案进行定量。简单地,100mg 的校准品或脑样本用10ml 的0.5 mg/ml 子囊霉素(内标)和300ml 的含有0.1% 甲酸和10mm 甲酸铵的溶液在95% 高效液相色谱级甲醇中溶解。超声波处理后,样品在15,000克下旋转离心,在23 ° c 下离心5min。上清液转移到1.5 ml 微滤管中,15,000 g 纺丝1分钟。40ml 最终提取物注入 LC/MS/MS。每个未知样品的雷帕霉素峰面积与子囊霉素标准峰面积的比值(响应比)与一个线性回归的校准器响应比值进行比较,以定量雷帕霉素。

4.7 Experimental design and statistical analysis

4.7实验设计及统计分析

Three experimental groups of male and female F344xBN rats were utilized: 16‐month‐old adults treated with vehicle chow (n = 10), 34‐month‐old aged rats treated with vehicle chow (n = 14), and 34‐month‐old aged rats treated with rapamycin in chow (n = 15). For all experiments, one‐way or two‐way ANOVAs were used to analyze differences among the group means on the outcome variables. Tukey’s post hoc comparison among all means was performed as indicated to clarify group differences. The data that support the findings of this study are available from the corresponding author upon reasonable request.

选用雄性和雌性 F344xBN 大鼠3个实验组: 16月龄成年大鼠10只,34月龄老年大鼠14只,34月龄老年大鼠15只。在所有实验中,采用单向和双向无变量分析组间平均值在结果变量上的差异。Tukey 的事后比较所有手段进行表明,以澄清群体差异。支持这项研究结果的数据可以从通讯作者合理的要求。

ACKNOWLEDGMENTS

鸣谢

We would like to acknowledge the technical expertise of Greg Friesenhahn for performing the blood rapamycin measurements using HPLC‐tandem MS. The authors would also like to acknowledge the following funding support: Alzheimer’s Association AARF‐17‐504221 (CEV), Ellison Medical Foundation AG‐NS‐0726‐10 (VG), US Department of Veterans Affairs Biomedical Laboratory Research and Development Service (VA‐BLRDS) Merit Award I01 BX002211‐01A2 (VG), NIH/NIA R01AG057964‐01 (VG), the Robert L. Bailey and daughter Lisa K. Bailey Alzheimer’s Fund in memory of Jo Nell Bailey (VG), William & Ella Owens Medical Research Foundation Grant (VG, SNA), the San Antonio Medical Foundation (VG), the JMR Barker Foundation (VG), NCATS/NIH UL1 TR002645 (VG), VA‐BLRDS 1 IK2 BX003798‐01A1 (SAH), NIH Biology of Aging Training Grant T32 AG‐021890 (CEV, SAH, JBJ), NIH/NIA K01AG040164 (ALL), NIH/NIA R01AG054459 (ALL), NIH/NIA R01AG062480 (ALL), American Federation for Aging Research Grant #A12474 (ALL), NIH/CTSA UL1TR0000117 (ALL), NIH/NIA RC2 AG036613, and P30 AG13319‐15S1 (SNA, KEF). The studies used the services of the San Antonio Nathan Shock Center of Excellence in the Biology of Aging (NIH/NIA 2 P30 AG013319‐21). The authors declare no competing financial interests.

我们要感谢 Greg Friesenhahn 的技术专长,他使用 HPLC-tandem ms 进行了血液雷帕霉素的测量。作者们还要感谢以下的资金支持: 老年痴呆症美国退伍军人事务部生物医学实验室研究与发展服务部(VA-BLRDS)荣誉奖 I01 BX002211-01A2(VG) ,NIH/NIA R01AG057964-01(VG) ,Robert l. Bailey 和女儿 Lisa k. Bailey’ Alzheimer’为了纪念 Jo Nell Bailey (VG)、 William & Ella Owens 医学研究基金会 Grant (VG,SNA)、圣安东尼奥医学基金会(VG)、 JMR Barker 基金会(VG) ,nCATS/NIH UL1 TR002645(VG) ,VA-BLRDS 1 IK2 BX003798-01A1(SAH) ,NIH 衰老生物学训练补助金 T32 AG-021890(CEV,SAH,JBJ) ,NIH/nia K01AG040164(ALL) ,NIH/R01AG054459(ALL) ,nIH/NIA R01AG062480(ALL) ,美国老龄化研究补助金 # a12474(ALL) ,NIH/CTSA ul1tr000117(ALL) ,NIH/NIA RC2 AG036613,P30 AG13319-15S1(SNA,KEF)。这些研究使用了圣安东尼奥内森休克卓越中心在衰老生物学方面的服务。两位作者声称没有相互竞争的经济利益。

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