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

摘要 苯二氮卓类(BZD)是一类广泛使用的镇静和抗焦虑药物，及其用法 随年龄增长(NIH新闻，2014)。而BZD对于治疗过度兴奋是非常有效的 精神障碍，它们可能会有严重的副作用，如运动障碍。绝大多数BZD老年用户 长期接受BZD治疗(NIH新闻，2014)，这会加剧运动障碍。这个 与其他年龄/性别人群相比，BZD的不良反应和长期使用在老年女性中更常见 组。然而，长期服用BZD(CBZD)如何损害老年女性的运动功能仍然存在 几乎是未知的。这项提议的目标是建立一种转基因小鼠模型，能够 确定cBZD导致女性受试者运动性衰老的机制。BZD与GABA结合- BZD受体复合体，从而产生中枢神经系统抑制作用。众所周知，BZD包括 地西潘、劳拉西潘和阿普唑仑与线粒体BZD受体(mBZD-R)结合，位于 线粒体膜。MBZD-R过度刺激损伤线粒体膜 线粒体发生呼吸作用，从而过量产生活性氧物种(ROS)。的约束性 认知障碍老年人小脑BZD to mBZD-R显著增加(Yasuno et 2012年)，表明mBZD-R在大脑老化中起作用。特别是，mBZD-R活动涉及亲- 凋亡蛋白p38：p38抑制剂可抑制mBZD-R配体诱导的细胞凋亡 等人，2004年)。由于ROS通过磷酸化激活p38，p38的凋亡作用在 缺乏直接清除ROS的17？雌二醇(E_2)的老年妇女。浦肯野细胞是主要的小脑 特别容易受到凋亡p38影响的神经元(见Approach，Guan等人，2005年)。浦肯野神经元 显示了p38的年龄相关性积累，这种积累可以被E2缓解(Jung等人，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- 与野生型小鼠相比，可引起运动障碍和线粒体呼吸抑制。这种保护作用 蒲肯野p38的下调在雌性小鼠中比雄性小鼠更明显，这表明雌性小鼠 荷尔蒙，尤其是雌二醇，可能在这种保护中发挥作用。这些观察表明，cBZD刺激了 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 和机动车老化。我们将通过实现具体目标来追求目标1：通过以下方式确定机制 其中cBZD会导致运动性衰老。我们将检验一个两步假设：1A)cBZD与mBZD-R AGE的结合- 依赖地抑制线粒体呼吸和1B)cBZD诱导浦肯野神经元凋亡和 在E2缺乏的年龄，通过ROS激活的p38导致的运动缺陷。这项拟议的研究绝对是至关重要的 因为预计它将建立一个坚实的模式和基础机制，这将是迈向 减轻服用cBZD的老年妇女的运动退化。

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.