A Novel Mouse Model to Study Motoric Aging Induced by Benzodiazepine Abuse

摘要 苯二氮卓类药物（BZD）是一类广泛使用的镇静和抗焦虑药物， 随着年龄的增长（NIH新闻，2014）。虽然BZD对治疗过度兴奋性 但是，如果他们患有疾病，他们可能会有严重的副作用，如运动缺陷。绝大多数老年BZD用户 长期接受BZD治疗（NIH新闻，2014），这会加剧运动缺陷。的 BZD的不良反应和长期使用在老年女性中比其他年龄/性别人群更多 组然而，慢性BZD（cBZD）使用如何损害老年女性的运动功能仍然存在 几乎无人知晓本提案的目的是建立一种能够 确定cBZD诱导女性受试者运动性衰老的机制。BZD结合GABA- BZD受体复合物，从而引发CNS抑制作用。众所周知，BZD包括 地西泮、劳拉西泮和阿普唑仑与线粒体BZD受体（mBZD-R）结合，位于 线粒体膜mBZD-R的过度刺激损伤线粒体膜， 发生线粒体呼吸，因此过度产生活性氧（ROS）。的结合 在患有认知障碍的老年人的小脑中，BZD至mBZD-R显著增加（Yasuno et 例如，2012），表明mBZD-R在大脑衰老中起作用。特别是，mBZD-R活性涉及前- 凋亡蛋白p38，使得p38抑制剂减弱由mBZD-R配体诱导的凋亡（Sutter et 例如，2004年）。由于ROS通过磷酸化激活p38，p38的凋亡作用在细胞中更严重。 缺乏直接清除ROS的17 β-雌二醇（E2）的老年妇女。浦肯野细胞是小脑的主要细胞 特别易受凋亡p38影响的神经元（参见Approach，Guan等人，2005年）。浦肯野神经元 显示了p38以被E2减轻方式的年龄依赖性积累（Jung等，2011年）。我们有 产生了浦肯野神经元中缺乏p38的小鼠，并观察到这些小鼠对cBZD- 诱导的运动缺陷和线粒体呼吸抑制。这种保护作用 浦肯野p38下调在雌性小鼠中比雄性小鼠更显著，这表明雌性小鼠 激素，尤其是E2，可能在保护中起作用。这些观察结果表明，cBZD引起 在E2缺乏的年龄，mBZD-R和p38之间的不利相互作用，导致浦肯野细胞凋亡 和运动性衰老我们将通过实现具体目标1来实现我们的目标： cBZD会导致运动性衰老我们将检验两步假设：1A）cBZD与mBZD-R的结合年龄- 依赖性抑制线粒体呼吸，1B）cBZD诱导浦肯野神经元凋亡， 在E2缺乏的年龄，通过ROS激活的p38导致运动缺陷。这项研究是绝对重要的 因为它有望建立一个坚实的模式和基础机制， 减轻接受cBZD的老年女性的运动功能恶化。

Abstract Benzodiazepines (BZDs) are a widely used class of sedative and anti-anxiety medications, and their usage increases with age (NIH news, 2014). While BZDs are powerfully effective for treating hyper-excitatory disorders, they can have serious side effects, such as motor deficits. The vast majority of elderly BZD users stays in BZD therapy for a prolonged period (NIH news, 2014), which exacerbates the motoric deficits. The adverse effects and long-term use of BZD are seen more in elderly women than other age/gender population groups. However, how chronic BZD (cBZD) usage impairs the motoric function of elderly women remains virtually unknown. The objective for this proposal is to establish a transgenic mouse model that is capable of identifying the mechanism by which cBZD induces motoric aging in female subjects. BZD binds to the GABA- BZD receptor complex, thereby eliciting CNS depressant effects. It is also well known that BZDs including diazepam, lorazepam, and alprazolam, bind to mitochondrial BZD receptors (mBZD-R), located in mitochondrial membranes. The excessive stimulation of mBZD-R damages mitochondrial membranes where mitochondrial respiration takes place, consequently overproducing reactive O2 species (ROS). The binding of BZD to mBZD-R was significantly increased in the cerebellum of the elderly with cognitive disorders (Yasuno et al., 2012), suggesting that mBZD-R plays a role in brain aging. In particular, mBZD-R activity involves pro- apoptotic protein p38 such that a p38 inhibitor attenuates the apoptosis induced by mBZD-R ligands (Sutter et al., 2004). Since ROS activates p38 by phosphorylation, the apoptotic effect of p38 would be more severe in elderly women who lack 17ß-estradiol (E2), which directly scavenges ROS. Purkinje cells are major cerebellar neurons that are particularly vulnerable to apoptotic p38 (see Approach, Guan et al., 2005). Purkinje neurons show an age-dependent accumulation of p38 in a manner that is mitigated by E2 (Jung et al., 2011). We have generated mice that lack p38 in Purkinje neurons, and observed that these mice are more resistant to cBZD- induced motoric deficit and mitochondrial respiratory suppression than wild-type mice. This protective effect of Purkinje p38 downregulation is more prominent in female mice than male mice, suggesting that a female hormone, especially E2, may play a role in that protection. These observations suggest that cBZD provokes an adverse interaction between the mBZD-R and p38 at an E2-deficient age, resulting in Purkinje apoptosis and motoric aging. We will pursue our objective by achieving Specific Aim 1: Determine the mechanism by which cBZD induces motoric aging. We will test a two-step hypothesis: 1A) cBZD's binding to mBZD-R age- dependently suppresses mitochondrial respiration and 1B) cBZD induces Purkinje neuronal apoptosis and motoric deficit through ROS-activated p38 at an E2-deficient age. The proposed research is absolutely critical because it is expected to establish a solid model and foundational mechanism that will be a first step toward lessening the motoric deterioration of elderly women receiving cBZD.